Pulsed electric field transfer of molecules to cells while in the body

ABSTRACT

Devices, systems and methods are provided for delivering molecules, particularly small molecules and/or macromolecules, to cells within the body, particularly to target cells which directly therapeutically benefit from the functionality of the molecules. Example molecules include DNA plasmids, RNAs (e.g. messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA), oligonucleotides, antisense oligonucleotides (ASO), proteins and/or materials which invoke genetic or epigenetic changes in the cellular behavior, to name a few. In such instances, the molecules are driven into the target cells with the use of pulsed electric fields (PEFs) which deliver the molecules through the cell wall of the target cells so that the desired genes are able to carry out the desired effect within the cells. Thus, the molecules are to be delivered to the desired location within the body at a desired time and in a desired concentration relative to the delivery of the pulsed electric fields for optimal outcomes. The molecules may be delivered to the body by a variety of methods, such as systemically, regionally and/or by direct injection to the area of the target cells. The pulsed electric field energy is delivered to the target cells in vivo and in situ which drives the molecules into the cells. Thus, the molecules are passed into the cells without the use of viruses or ex vivo methods, such as in vitro electroporation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No. PCT/US21/44469, filed Aug. 4, 2021, which claims priority to and the benefit of U.S. Provisional Patent Application No. 63/061,114, filed Aug. 4, 2020; U.S. Provisional Patent Application No. 63/061,091, filed Aug. 4, 2020; and U.S. Provisional Patent Application No. 63/209,335, filed Jun. 10, 2021, for which the disclosures of all of the foregoing applications are incorporated herein by reference in their entireties.

BACKGROUND

Genes create a nucleotide-based storage system of instructions critical to the existence, function, and replication of cells. Each gene is comprised of a distinct sequence of nucleotides (small segment of DNA) that codes for a specific protein which has a particular function in the organism. Enzymes transcribe the segment of DNA by building a single-stranded molecule of RNA. Like DNA, RNA is a strand of nucleotides. This transcribed RNA (i.e. messenger RNA or mRNA) leaves the nucleus of the cell and travels into the cytoplasm where ribosomes translate the mRNA to make the protein specified by the DNA. These proteins are the building blocks of life.

However, the DNA replication processes are not perfect, resulting in the generation of mutations over the course of replication. Oftentimes, these mutations are inconsequential, or can give rise to improved or reduced efficacy of protein formation. However, at other times, mutations to genes can hinder the proper function of the protein. This can give rise to many diseases, such as cancer or genetic disorders such as hemophilia, cystic fibrosis and many more. When the gene mutations are hard-encoded in a parent's sex chromosome, these traits can be inherited. At times, both parents must have damaged genes for a disease to arise, such as sickle-cell anemia. At other times, a damaged gene on a single chromosome can cause disease, such as conversion from a proto-oncogene to an oncogene, resulting in uncontrolled cell division that may lead to cancer formation.

A number of gene therapies have been developed in recent decades in attempts to correct or improve the production of proteins that have been affected by such mutations. In some instances, gene therapies implant the correct version of a gene so that the proper protein is produced. For example, genes that encode for clotting factors have been introduced to patients suffering from hemophilia. In other instances, the gene therapy invokes the upregulation of expression of specific molecules which bolster the cellular output of a correct protein, such as invoking cells to produce immunostimulant molecules for treating diseases such as cancer.

However, introduction of genetic material into cells has its challenges. Unlike many other biological molecules and therapeutic compounds, DNA sequences are not designed to travel across cell membranes. Further, they lack endocytotic factors that would induce alternate mechanisms of transport into cells. Therefore, all gene therapies require a combination of at least two features: the therapeutic encoded molecule (DNA sequence, gene, siRNA, etc) and a route of administration into the targeted cells. Several approaches to accomplish this have been developed.

Viral Vectors

Viruses, including lentiviruses and adeno-associated viruses, are well suited to the delivery of genetic material. Viruses, by their nature, rely on the delivery of their viral DNA or RNA into host cells for the replication of viral materials, thus making them very potent candidates for gene therapy. Delivery of genes, or other genetic material, by a vector is termed transduction and the infected cells are described as transduced.

When utilizing viruses for transduction, the genes in the virus that cause disease are removed and replaced with the desired genes that encode the desired effect (e.g. insulin production in the case of diabetics). This is achieved in such a way that the genes which allow the virus to infect the host are left intact. The desired genes are introduced to the virus with the use of a plasmid. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA. They are most commonly found as small circular, double-stranded DNA molecules in bacteria, however artificially constructed plasmids are used as vectors in genetic engineering. A gene of interest is typically inserted into a vector through different cloning methods such as restriction enzyme ligation, ligation independent cloning, Gateway cloning and Gibson assembly. The cloning method chosen depends on the plasmid. Following the cloning process, the reconstructed vector containing a gene of interest is generated.

In some instances, the plasmid itself does not replicate and the potency of the gene effect may decay over time as the plasmid degrades, the host cell divides, or the host cell dies. In other instances, the genetic material merges into the host cell's DNA. While durable integration of genetic material into the host's DNA using viral vectors has a more potent effect, it may also introduce risks, that are both known and unknown, and cannot be reversed if there are issues with this gene. Inactivation of the disease-inducing aspects of viruses is costly and may be incomplete, resulting in infection risks to gene therapy patients. Viruses may also migrate beyond targeted-site gene therapies. Viruses can usually infect more than one type of cell. Thus, when viral vectors are used to carry genes into the body of cancer patients, they might infect healthy cells as well as cancer cells. Likewise, the new gene might be inserted in the wrong location in the DNA during integration, possibly causing harmful mutations to the DNA or even cancer. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 4 of 20 patients. Further, the deliberate introduction of viruses, pathogenic or otherwise, invokes the host's immune system. The immune system may depreciate the effectiveness of the virus gene therapy by preventing transduction. Furthermore, this induction may cause severe side effects for the patients, and a number of deaths from viral-based gene therapies have been reported. Ultimately, viral-based gene therapies are costly, risky, and have varying degrees of efficacy, particularly for target-site gene therapies.

Electroporation-Mediated Gene Transfer

Electroporation has been known for over three decades as a method to disrupt cell membrane integrity and result in macromolecule uptake for cells in suspension. Typically, electroporation is achieved with the use of an electroporation cuvette 10 and an electroporator 12, such as illustrated in FIGS. 1A-1B. Referring to FIG. 1A, the cuvette 10 is comprised of glass or plastic and includes a reservoir 14 for receiving a suspension of cells C. A pair of electrode plates 16 a, 16 b are disposed an established distance apart on opposite sides of the reservoir 14. Cells C are suspended in media that is pipetted into the cuvette 10. The cuvette 10 is then placed in the electroporator 12, as illustrated in FIG. 1B. The electroporator 12 contains a capacitor which is charged with a high voltage and when directed, discharges the stored current within the capacitor into the sample of cells C in the cuvette 10. The capacitance discharge circuit generates an electrical pulse having a peak voltage and an exponential decay waveform. The electric field strength is the voltage applied between the electrode plates 16 a, 16 b and can be described by electric field strength=voltage/distance where distance is the distance between the plates 16A, 16 b. The electric field strength and the size of the cells determine the voltage drop across each cell and therefore the voltage effect in electroporation. Thus, protocols are typically developed specific to cell type. Likewise, parameters, such as field strength, time constant and width of decay pulse, may be altered for each protocol. Thus, the in vitro environment permits very precise control of transfection conditions, permitting high levels of gene delivery without killing too large of a proportion of the cells.

Overall, transfection in vitro has a high reliability for generating fundamental understanding of gene effects or producing transgenic experimental animal models. However, it has difficulty in implementing viable clinical gene therapies. The need for transfecting cells in culture requires several additional steps in translating to a therapy for patients. Most often, an apheresis-based approach is used to deliver a gene therapy. This involves sampling a patient's cells through a blood draw or biopsy. The targeted cell population is then isolated and concentrated. Oftentimes, the cell population is also amplified to produce a meaningful population size of transfected cells. Once the cells have been prepared, they are then electroporated with the genetic therapeutic molecule. Following transfection, the cells are then redistributed at the necessary site in the patient, such as the blood or the target location in the targeted organ. These steps add substantial burden, cost, invasiveness, time, and risk to delivering a gene therapy. Overall, these numerous and substantial drawbacks have prevented widespread adoption of pulsed electric field-based gene therapies for most potential therapeutic options.

Overall, although genetic-based therapies offer a wide array of potential applications, the clinical translation of these therapies remains in its infancy, mainly due to the limitations of the gene delivery administration routes, which are costly, time-consuming, risky, ineffective, or inapplicable for certain disease states. Therefore, improvements in gene therapy are desired. Such treatments should be safe, effective, and lead to reduced complications. Such treatments should also be applicable to therapies involving transfer of other types of molecules to cells, particularly macromolecules. At least some of these objectives will be met by the systems, devices and methods described herein.

SUMMARY OF THE INVENTION

Described herein are embodiments of apparatuses, systems and methods for treating target tissue in the body. Likewise, the invention relates to the following numbered clauses:

1. A system for transferring molecules to target tissue cells within a body of a patient comprising:

-   -   an energy delivery device having at least one energy delivery         body configured to be positioned near the target tissue cells         within the body; and     -   a generator in electrical communication with the at least one         energy delivery body, wherein the generator includes at least         one energy delivery algorithm configured to provide an electric         signal deliverable to the at least one energy delivery body so         as transmit pulsed electric field energy which causes at least         one of the molecules to enter at least one cell of the target         tissue cells.

2. A system as in claim 1, wherein the electric signal comprises a series of pulses, wherein the series of pulses includes at least one pulse having a positive amplitude and at least one pulse of having a negative amplitude.

3. A system as in claim 2, wherein together the series of pulses have a balance of charge from positive amplitude on-time and negative amplitude on-time.

4. A system as in any of claims 2-3, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid muscle stimulation within the body.

5. A system as in any of claims 2-4, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid ablation of the target tissue cells.

6. A system as in any of the above claims, wherein the electric signal comprises a series of pulses including at least one pulse that differs in amplitude or pulse width.

7. A system as in any of the above claims, wherein the electric signal comprises a series of pulses wherein at least one pulse has a voltage in a range of 10-500V.

8. A system as in claim any of the above claims, wherein the electric signal comprises a series of pulses wherein at least one pulse has a pulse duration in a range of 0.5-200 ms.

9. A system as in any of the above claims, wherein the electric signal comprises a series of pulses having at least one interpulse delay in a range of 10 ms-10 s.

10. A system as in any of the above claims, wherein the electric signal comprises a series of pulses having 1-100 pulses.

11. A system as in claim any of the above claims, wherein the electric signal comprises a series of pulses together having a duration of 0.5 to 500 ms.

12. A system as in claim 1, wherein the electric signal comprises a series of pulses wherein at least one pulse has a base pulse width of sufficient length to cause muscle stimulation in the body and wherein the at least one pulse includes a plurality of sectional-pulses having sectional-delays therebetween sufficient to at least reduce the muscle stimulation.

13. A system as in claim 12, wherein the base pulse width is in the range of 0.01-50,000 μs.

14. A system as in claim 13, wherein the at least one pulse has a pulse width of 1-100,000 μs due to inclusion of the plurality of sectional pulses and sectional delays to the base pulse width.

15. A system as in claim 14, wherein the plurality of sectional-pulses comprises up to sectional-pulses

16. A system as in claim 15, wherein each sectional-pulse has a duration in a range of 0.05-5 μs.

17. A system as in claim 15, wherein each sectional delay has a duration in a range of 0.001-10 μs.

18. A system as in claim 14, wherein each sectional-pulse has a duration in a range of 0.05-50 μs.

19. A system as in claim 14, wherein each sectional-delay has a duration in a range of 0.001-100 ms.

20. A system as in claim 14, wherein the at least one sectional-pulse has an on-time that does not exceed 10 μs.

21. A system as in claim 12, wherein the at least one pulse comprises at least two pulses separated by a delay of 10 ms-10 s.

22. A system as in claim 21, wherein the at least two pulses have opposite polarity.

23. A system as in claim 22, wherein each of the pulses in the series of pulses has a base pulse width of sufficient length to cause muscle stimulation in the body and wherein each of the pulses in the series of pulses includes a plurality of sectional-pulses having sectional-delays therebetween sufficient to at least reduce the muscle stimulation.

24. A system as in claim 23, wherein together the pulses in the series of pulses have a balance of charge from positive amplitude on-time and negative amplitude on-time.

25. A system as in any of claim 12-24, wherein the at least one pulse has a voltage in a range of 10-250V.

26. A system as in claim 1, wherein the electric signal comprises a series of biphasic pulses wherein at least one biphasic pulse has a cycle length of 0.01-10 μs and wherein the at least one biphasic pulse includes a plurality of sectional-pulses having sectional delays therebetween.

27. A system as in claim 26, wherein the plurality of sectional-pulses comprises up to 10,000 sectional-pulses.

28. A system as in claim 27, wherein each sectional pulse has a duration in a range of 0.004-0.4 μs.

29. A system as in claim 28, wherein each sectional delay has a duration in a range of 10-10,000 μs.

30. A system as in claim 28, wherein each sectional pulse has a duration in a range of 0.05-50 μs.

31. A system as in claim 26, wherein each sectional delay has a duration in a range of 0.001-100 ms.

32. A system as in claim 26, wherein the at least one sectional pulse has an on-time that does not exceed 10 μs.

33. A system as in claim 26, wherein the series of biphasic pulses comprises up to 1000 cycles.

34. A system as in any of claims 26-33, wherein the series of biphasic pulses are grouped into packets having inter-packet delays therebetween.

35. A system as in in any of claims 26-34, wherein the at least one biphasic pulse has a voltage in a range of 500-2000V.

36. A system as in claim 1, wherein the electric signal comprises a series of pulses, wherein the series of pulses includes at least one high voltage, high frequency pulse followed by at least one low voltage, low frequency pulse, wherein high voltage is in a range of 100-1000V, high frequency has a pulse width of 50 ns-1 ms, low voltage is in a range of 5-100V, and low frequency has a pulse width of 1 ms-50 ms.

37. A system as in claim 36, wherein the at least one low voltage, low frequency pulse comprises at least two low voltage, low frequency pulses having opposite polarity.

38. A system as in claim 36, wherein the at least one low voltage, low frequency pulse comprises a DC waveform.

39. A system as in claim 36, wherein the at least one high voltage, high frequency pulse comprises a biphasic waveform.

40. A system as in claim 36, wherein the at least one high voltage, high frequency pulse is separated from the at least one low voltage, low frequency pulse by a delay of 100 μs-2 seconds.

41. A system as in any of the above claims, wherein the electric signal comprises a series of pulses wherein at least one pulse includes at least one spike.

42. A system as in claim 41, wherein the at least one pulse has a primary voltage and the at least one spike comprises a plurality of spikes formed by oscillations around the primary voltage.

43. A system as in claim 42, wherein the oscillations are in a range of 1-25% of the primary voltage.

44. A system as in claim 43, wherein the oscillations are in a range of 1-10% of the primary voltage.

45. A system as in any of claims 41-44, wherein the at least one pulse includes a plurality of spikes which are not evenly distributed along the at least one pulse.

46. A system as in any of claims 41-45, wherein the series of pulses are biphasic.

47. A system as in any of the above claims, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom.

48. A system as in claim 47, wherein the energy delivery device is configured for delivery of the molecules through at least one of the one or more tines.

49. A system as in claim 48, wherein the energy delivery device is configured for delivery of different molecules through at least one of the one or more tines in comparison to at least another of the one or more tines.

50. A system as in any of claims 47-49, wherein the at least one energy delivery body comprises at least one of the one or more tines.

51. A system as in claim 50, wherein the at least one energy delivery body comprises at least two of the one or more tines which are individually energizable.

52. A system as in any of the above claims, wherein the at least one energy delivery body comprises a basket-shaped electrode.

53. A system as in any of the above claims, wherein the energy delivery device comprises an elongate shaft and wherein the at least one energy delivery body comprises at least two protrusions, each protrusion extending radially outwardly from the elongate shaft.

54. A system as in any of the above claims, wherein the generator includes at least one other energy delivery algorithm configured to provide another electric signal deliverable to the at least one energy delivery body so as to transmit conditioning energy which induces extravasation of fluid from the body within a localized area of the target tissue cells.

55. A system as in claim 54, wherein the extravasation is from vasculature of the body of the patient.

56. A system as any of claims 54-55, wherein the localized area comprises interstitial spaces around the target tissue cells.

57. A system as in any of claims 54-56, wherein the molecules are delivered to vasculature of the body and wherein the induced extravasation delivers the molecules from the vasculature to the localized area.

58. A system as in any of claims 54-57, wherein the another electric signal comprises a plurality of monophasic pulses each having a duration exceeding 500 microseconds.

59. A system as in any of claims 54-57, wherein the another electric signal comprises a plurality of pulses wherein at least one of the plurality of pulses has a positive amplitude and wherein at least one of the plurality of pulses as a negative amplitude.

60. A system as in claim 59, wherein the each of the plurality of pulses has a duration exceeding 500 microseconds.

61. A system as in any of claims 54-60, wherein the energy delivery device includes at least one pressure sensor.

62. A system as in claim 61, wherein the at least one pressure sensor is configured to monitor effects of the extravasation and provide sensor feedback data.

63. A system as in claim 62, wherein the system includes a mechanism to provide the sensor feedback data or information based on the sensor feedback data to a user.

64. A system as in claim 62, wherein the generator includes a processor configured to modify the at least one energy delivery algorithm or switch to a different energy delivery algorithm based on the sensor feedback data so as to transmit energy which adjusts inducement of extravasation.

65. A system as in any of claims 1-60, wherein the system includes at least one sensor.

66. A system as in claim 65, wherein the at least one sensor comprises a sensor that monitors pressure, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties, coherence, echogenicity, fluorescence, electrical permittivity, light permittivity, and/or conductance.

67. A system as in any of the above claims, wherein the generator includes an additional energy delivery algorithm configured to provide an ablation electric signal deliverable to the at least one energy delivery body so as to transmit ablation energy which causes at least one of the target tissue cells to die.

68. A system as in any of the above claims, wherein the wherein the at least one molecule comprises a small molecule and/or a macromolecule.

69. A system as in any of the above claims, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomer molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolysis targeting chimera, Zinc Finger Nucleases or Transcription Activator-Like Effector Nucleases, a protein and/or material which invokes genetic or epigenetic changes in cellular behavior.

70. A system as in any of the above claims, wherein the at least one molecule comprises at least one gene larger than 5 kb.

71. A system as in claim 70, wherein the at least one molecule comprises at least one gene larger than 10 kb.

72. A system as in any of the above claims, wherein the target tissue cells comprise cells of a retina.

73. A system as in claim 72, wherein the cells of the retina include cells of a Layer of Rods and Cones and/or cells of a retinal pigment epithelium.

74. A system as in any of claims 1-71, wherein the target tissue cells comprise cells of a bone marrow.

75. A system as in any of claims 1-71, wherein the target tissue cells comprise cells of a digestive system, including a liver, a pancreas, intestines and/or colon.

76. A system as in any of claims 1-71, wherein the target tissue cells comprise cells of a heart.

77. A system for treating target tissue cells within a body of a patient comprising:

-   -   an energy delivery device comprising a shaft having one or more         tines extending therefrom, wherein the one or more tines         includes a first energy delivery body and a second energy         delivery body, wherein the one or more tines are configured to         be positioned near the target tissue cells within the body of         the patient;     -   a generator in electrical communication with the first and         second energy delivery bodies, wherein the generator includes at         least a first energy delivery algorithm configured to provide a         first electric signal deliverable to the first energy delivery         body and at least a second energy delivery algorithm configured         to provide a second electric signal deliverable to the second         energy delivery body.

78. A system as in claim 77, wherein the first electric signal generates energy that causes transfer of at least one molecule to at least one of the target tissue cells.

79. A system as in any of claims 77-78, wherein the second electric signal generates energy that causes ablation of at least one target tissue cell.

80. A system as in any of claims 77-79, wherein at least one of the one or more tines extends distally from a distal end of the shaft along its longitudinal axis, and wherein the second energy delivery body is disposed therealong.

81. A system as in any of claims 77-80, wherein at least one of the one or more tines extends radially from the shaft, and wherein the first energy delivery body is disposed therealong.

82. A system as in claim 77, wherein the one or more tines are configured to allow ablation of a first area of the target tissue cells and allow transfer molecules to second area of the target tissue cells.

83. A system as in claim 82, wherein the second area at least partially surrounds the first area.

84. A system as in any of claims 77-83, wherein energy delivery device is configured so that at least one of the one or more tines is able to deliver a plurality of molecules.

85. A system as in claim 84, wherein the energy delivery device is configured for delivery of different molecules through at least one of the one or more tines in comparison to at least another of the one or more tines.

86. A system as in any of claims 77-85, wherein at least one of the one or more tines is adjustably extendable.

87. A method of transferring molecules to cells of target tissue within a body of a patient comprising:

-   -   delivering a plurality of molecules to the body of the patient;     -   positioning at least one energy delivery body of an energy         delivery device within sufficient range of target tissue so as         to receive pulsed electric field energy delivered therefrom; and         delivering the pulsed electric field energy to the at least one         energy delivery body so as to transmit the pulsed electric field         energy to the target tissue in a manner which causes at least         one of the molecules to enter at least one of the cells of the         target tissue.

88. A method as in claim 87, wherein the target tissue cells directly therapeutically benefit from the functionality of the molecules.

89. A method as in claim 88, wherein directly therapeutically benefit comprises treatment of a disorder.

90. A method as in claim 89, wherein the disorder comprises a genetic disorder.

91. A method as in any of claims 87-90, wherein the at least one molecule comprises a small molecule and/or a macromolecule.

92. A method as in any of claims 87-91, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomer molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolysis targeting chimera, Zinc Finger Nucleases or Transcription Activator-Like Effector Nucleases, a protein and/or material which invokes genetic or epigenetic changes in cellular behavior.

93. A method as in any of claims 87-92, wherein the at least one molecule comprises at least one gene larger than 5 kb.

94. A method as in claim 93, wherein the at least one molecule comprises at least one gene larger than 10 kb.

95. A method as in any of claims 87-94, wherein delivering the plurality of molecules to the body comprises delivering the molecules intravenously to the body.

96. A method as in claim 87-94, wherein delivering the plurality of molecules to the body comprises delivering the molecules intravenously to the body and locally to the target tissue.

97. A method as in any of claims 87-96, wherein delivering the plurality of molecules occurs at least prior to delivering the energy.

98. A method as in any of claims 87-97, wherein delivering the plurality of molecules comprises delivering the plurality of molecules to multiple locations within or near the target tissue.

99. A method as in claim 98, wherein the multiple locations are within 0.5 mm-5 cm of the at least one cell of the target tissue.

100. A method as in any of claims 98-99, wherein delivering the plurality of molecules at multiple locations comprises delivering different concentrations of molecule solution, different volumes of molecule solution and/or different types of molecule solution to one or more of the multiple locations.

101. A method as in any of claims 87-99, wherein delivering the plurality of molecules to multiple locations includes delivering different molecules to at least one of the multiple locations in comparison to another of the multiple locations.

102. A method as in any of claims 87-101, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom, and wherein delivering the plurality of molecules at multiple locations is achieved by delivering molecules through one or more of the one or more tines.

103. A method as in claim 102, wherein the delivering the plurality of molecules comprises delivering different molecules through at least one of the one or more tines in comparison to a least another of the one or more tines.

104. A method as in claim 102, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom, and wherein the at least one energy delivery body comprises at least one of the one or more tines, and wherein delivering the energy comprises energizing at least one of the plurality of tines.

105. A method as in claim 104, wherein energizing at least one of the plurality of tines comprises individually energizing at least one of the plurality of tines while at least one of the plurality of tines is not energized.

106. A method as in any of claims 87-105, further comprising inducing extravasation of fluid within a localized area in the body.

107. A method as in claim 106, wherein inducing extravasation occurs prior to delivering energy.

108. A method as in any of claims 106-107, wherein the extravasation increases delivery of molecules to the target tissue.

109. A method as in any of claims 106-108, wherein delivering the plurality of molecules comprises delivering the plurality of molecules to vasculature of the body and wherein the extravasation is from the vasculature.

110. A method as in claim 106, wherein inducing extravasation comprises delivering conditioning energy to the at least one energy delivery body so as to transmit the conditioning energy to the body in a manner which induces extravasation of fluid from the body within a localized area of the target tissue.

111. A method as in claim 110, wherein the conditioning energy is from another electric signal comprising a plurality of monophasic pulses each having a duration exceeding 500 microseconds.

112. A method as in any of claims 110-111, wherein the conditioning energy comprises PEF energy.

113. A method as in claim 87, further comprising delivering conditioning energy to the target tissue prior to delivering the pulsed electric field energy.

114. A method as in claim 113, wherein the conditioning increases cellular resistance of the target tissue to eventual cell death.

115. A method as in any of claims 87-114, further comprising pre-warming the target tissue prior to delivering the energy.

116. A method as in claim 115, wherein pre-warming the target tissue comprises delivering conditioning energy to the target tissue.

117. A method as in any of claims 87-116, wherein the pulsed electric field energy is from an electric signal comprising a series of pulses, wherein the series of pulses includes at least one pulse having a positive amplitude and at least one pulse of having a negative amplitude.

118. A method as in claim 117, wherein together the series of pulses have a balance of charge from positive amplitude on-time and negative amplitude on-time.

119. A method as in claim 117, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid muscle stimulation within the body.

120. A method as in claim 117, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid ablation of the target tissue cells.

121. A system for treating a portion of an eye of a patient comprising:

-   -   an instrument having at least one electrode body configured to         be positioned in, on or near the eye; and     -   a generator in electrical communication with the at least one         electrode body, wherein the generator includes at least one         energy delivery algorithm configured to provide an electric         signal of pulsed electric field energy deliverable to the at         least one electrode body so as to cause at least one molecule to         enter a cell of the eye.

122. A system as in claim 121, wherein the instrument comprises a shaft having a distal end, wherein the at least one electrode body is disposed near the distal end of the shaft.

123. A system as in any of claims 121-122, wherein the shaft is configured to be insertable into a vitreous space of the eye.

124. A system as in any of claims 121-123, wherein at least one of the at least one electrode body is configured to be insertable into a subretinal bleb within the eye.

125. A system as in any of claims 121-123, wherein at least one of the at least one electrode body is configured to be insertable into a suprachoroidal space within the eye.

126. A system as in any of claims 121-123, wherein the at least one electrode body comprises a bipolar pair of electrode bodies.

127. A system as in claim 126, wherein the bipolar pair of electrode bodies comprises a first electrode body disposed so as to be positionable within a subretinal bleb while a second electrode body is disposed so as to be positionable outside of the subretinal bleb.

128. A system as in claim 126, wherein the bipolar pair of electrode bodies comprises a first electrode body disposed so as to be positionable within a suprachoroidal space while a second electrode body is disposed so as to be positionable outside of the suprachoroidal space.

129. A system as in claim 126, wherein the bipolar pair of electrode bodies comprises a first electrode body disposed so as to be positionable within a vitreous space while a second electrode body is disposed so as to be positionable outside of the vitreous space.

130. A system as in claim 126, wherein the bipolar pair of electrode bodies comprises a first electrode body disposed so as to be positionable at a first location and a second electrode body disposed so as to be positionable at a second location, wherein a portion of a retina is disposed between the first and second locations.

131. A system as in claim 126, wherein polarities of the pair of bipolar of electrode bodies are reversible.

132. A system as in any of claims 121-125, further comprising a return electrode configured to be positioned at a distance from at least one of the at least one electrode body so that the at least one electrode body functions in a monopolar fashion.

133. A system as in claim 132, wherein the return electrode is configured to be positioned against or near an outer surface of the eye.

134. A system as in claim 133, wherein the return electrode comprises an electroretinography electrode, a speculum, a contact lens, or a tweezertrode electrode.

135. A system as in claim 132, wherein the return electrode is configured to be positioned at least partially in a retrobulbar space.

136. A system as in claim 132, wherein the return electrode is configured to be positioned at least partially in a suprachoroidal space.

137. A system as in any of claims 121-136, wherein the instrument includes a lumen for delivery of a fluid.

138. A system as in claim 137, wherein the instrument includes at least one outlet in fluid communication with the lumen so that the fluid is deliverable near at least one of the electrode body.

139. A system as in claim 137, further comprising the fluid, wherein the fluid comprises small molecules and/or macromolecules.

140. A system as in any of claims 121-139, wherein the at least one molecule comprises a small molecule and/or a macromolecule.

141. A system as in any of claims 121-139, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomer molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolysis targeting chimera, Zinc Finger Nucleases or Transcription Activator-Like Effector Nucleases, a protein and/or material which invokes genetic or epigenetic changes in cellular behavior.

142. A system as in any of claims 121-141, wherein the at least one molecule comprises at least one gene larger than 5 kb.

143. A system as in claim 142, wherein the at least one molecule comprises at least one gene larger than 10 kb.

144. A system as in any of claims 121-143, wherein the cell is disposed within a retina of the eye.

145. A system as in claim 144, wherein the cell is disposed within a Layer of Rods and Cones of the retina.

146. A system as in claim 144, wherein the cell is disposed within a retinal pigment epithelium of the retina.

147. A method for treating an eye of a patient comprising:

-   -   positioning at least one electrode body in or near the eye;     -   introducing at least one molecule to a portion of the eye; and     -   delivering pulsed electric field energy to the at least one         electrode body so as to cause at least one of the at least one         molecule to enter a cell within the eye.

148. A method as in claim 147, wherein the pulsed electric field energy comprises a series of monophasic pulses.

149. A method as in claim 148, wherein the series of monophasic pulses comprises a series of low voltage pulses.

150. A method as in claim 147, wherein the pulsed electric field energy comprises a series of biphasic pulses.

151. A method as in any of claims 147-150, wherein positioning the at least one electrode body comprises positioning at least one of the at least one electrode body into a vitreous space of the eye.

152. A method as in any of claims 147-150, wherein positioning the at least one electrode body comprises positioning at least one of the at least one electrode body into a subretinal bleb within the eye.

153. A method as in any of claims 147-150, wherein positioning the at least one electrode body comprises positioning at least one of the at least one electrode body into a suprachoroidal space within the eye.

154. A method as in claim 147, wherein the at least one electrode body comprises a bipolar pair of electrode bodies and wherein positioning the at least one electrode body comprises positioning a first electrode body within a subretinal bleb and positioning a second electrode body outside of the subretinal bleb.

155. A method as in claim 147, wherein the at least one electrode body comprises a bipolar pair of electrode bodies and wherein positioning the at least one electrode body comprises positioning a first electrode body within a suprachoroidal space and positioning a second electrode body outside of the suprachoroidal space.

156. A method as in claim 147, wherein the at least one electrode body comprises a bipolar pair of electrode bodies and wherein positioning the at least one electrode body comprises positioning a first electrode body within a vitreous space and positioning a second electrode body outside of the vitreous space.

157. A method as in claim 147, wherein the at least one electrode body comprises a bipolar pair of electrode bodies and wherein positioning the at least one electrode body comprises positioning a first electrode body at a first location and a second electrode body at a second location, wherein a portion of a retina is disposed between the first and second locations.

158. A method as in any of claims 154-157, further comprising reversing polarity of the bipolar pair.

159. A method as in claim 147, wherein the cell resides within a layer of a retina of the eye and further comprising delivering energy to the at least one electrode body so as to cause at least one of the at least one molecule to enter a cell within a different layer of the retina.

160. A method as in claim 147, further comprising positioning a return electrode at a distance from at least one of the at least one electrode body so that the at least one electrode body is able to function in a monopolar fashion.

161. A method as in claim 160, wherein positioning the return electrode comprises positioning the return electrode against or near an outer surface of the eye.

162. A method as in claim 161, wherein the return electrode comprises an electroretinography electrode, a speculum, a contact lens, or a tweezertrode electrode.

163. A method as in claim 160, wherein positioning the return electrode comprises positioning the return electrode at least partially in a retrobulbar space.

164. A method as in claim 160, wherein positioning the return electrode comprises positioning the return electrode at least partially in a suprachoroidal space.

165. A method as in any of claims 147-164, wherein the at least one molecule comprises at least one small molecule and/or macromolecule.

166. A method as in any of claims 147-164, wherein the at least one molecule comprises a plasmid, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomer molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a protein and/or material which invokes genetic or epigenetic changes in cellular behavior.

167. A method as in any of claims 147-164, wherein the at least one molecule comprises a synthetic DNA vector.

168. A method as in any of claims 147-167, wherein the cell is disposed within a retina of the eye.

169. A method as in claim 168, wherein the cell is disposed within a Layer of Rods and Cones of the retina.

170. A method as in claim 168, wherein the cell is disposed within a retinal pigment epithelium of the retina.

These and other embodiments are described in further detail in the following description related to the appended drawing figures.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.

FIGS. 1A-1B illustrate an example of a conventional in vitro electroporation system.

FIG. 2 illustrates an embodiment of a transfer system.

FIG. 3A illustrates direct injection of plasmid to a target tissue through a needle.

FIG. 3B illustrates delivery of energy from an energy delivery device inserted in place in the needle of FIG. 3A.

FIG. 4A illustrates direct injection of plasmid to a target tissue through an energy delivery body.

FIG. 4B illustrates delivery of energy from the energy delivery body of FIG. 4A.

FIG. 5 illustrates plasmid delivered regionally while energy is delivered locally, and optionally plasmid is delivered locally as well.

FIG. 6 illustrates an energy delivery device comprising a shaft having an energy delivery body near its distal end, wherein the energy delivery body comprises a plurality of tines.

FIG. 7 illustrates an energy delivery device comprising an energy delivery body having a basket shape configured for treating target tissue endoluminally.

FIG. 8 illustrates another embodiment of an energy delivery device comprising an energy delivery body having a shape configured for treating target tissue endoluminally, wherein the energy delivery body comprises at least two protrusions, each protrusion extending radially outwardly so as to contact an inner luminal wall.

FIG. 9 illustrates another embodiment of an energy delivery device comprising an energy delivery body having a shape configured for treating target tissue endoluminally, wherein the energy delivery body comprises an expandable member, such as an inflatable balloon, having an electrode mounted thereon or incorporated therein.

FIG. 10 illustrates an embodiment of an energy delivery device 102 wherein the delivery body 108 has a finger-tip shape configured to contact an inner lumen wall.

FIGS. 11A-11C illustrate various stages of an embodiment of an extravasation procedure.

FIGS. 12A-12B illustrate an example waveforms of pulsed electric field (PEF) energy provided by an energy delivery algorithm of the generator used for inducing extravasation.

FIG. 13 illustrates data resulting from various types of delivery of enhanced green fluorescent protein (EGFP)-encoding plasmid DNA with PEF energy.

FIG. 14 illustrates the data of FIG. 13 as average values.

FIGS. 15A-15C illustrate example waveforms based on DC currents provided by an energy delivery algorithm.

FIG. 16 illustrates an embodiment of a specialized waveform that alternates in polarity, wherein the pulses are “chopped”, partitioned or sectioned into an array of pulses of the same phase.

FIG. 17 illustrates an embodiment of a biphasic waveform, wherein the pulses are “chopped”, partitioned or sectioned into an array of pulses of the same phase.

FIGS. 18A-18C illustrates embodiments of a waveforms having a combination of high and short pulses combined with low and long pulses.

FIG. 19 illustrates packet comprising a series of pulses grouped into packets.

FIG. 20 illustrates a packet comprising a series of biphasic pulses or cycles, each biphasic pulse having a switch time delay.

FIGS. 21-22 illustrate embodiments of waveforms having a series of high voltage, high frequency pulses followed by a series of low voltage, low frequency pulses wherein the combination assists in transferring molecules to target cells.

FIG. 23 illustrates another embodiment of a pulse waveform.

FIGS. 24A-24C illustrate additional embodiments of waveforms provided by the algorithms 152, wherein pulses include rapid oscillations of high frequency.

FIGS. 25A-25C illustrate pressure sensors disposed at a variety of locations along a distal end of an energy delivery device.

FIG. 26 illustrates an embodiment of an endoluminal energy delivery device comprising an energy delivery body and appropriate maneuverability to facilitate placement and energy delivery to the walls of organs, such as the heart, directly.

FIG. 27 illustrates the placement of the coronary arteries in relation to the aorta and the position of the energy delivery device of FIG. 26 .

FIG. 28 illustrates a cross-sectional portion of a retina near an optic nerve.

FIG. 29 illustrates an embodiment of an energy delivery system configured for delivery of energy to portions of the eye.

FIG. 30 illustrates an embodiment of a procedure for creating a subretinal bleb.

FIG. 31 illustrates an embodiment of positioning of an energy delivery device into the eye so that an electrode body is positioned within the subretinal bleb.

FIGS. 32A-32B illustrate molecules that have entered the retinal pigment epithelium upon application of pulsed electric field energy.

FIG. 33 illustrates an embodiment wherein molecules are delivered to the subretinal bleb and an electrode body is positioned within the subretinal bleb for monopolar energy delivery thereto.

FIG. 34 illustrates an embodiment wherein molecules are delivered to the subretinal bleb and an electrode body is positioned within the vitreous space of the eye for monopolar delivery thereto.

FIG. 35 illustrates an embodiment wherein molecules are delivered to the vitreous space and an electrode body is also positioned in the vitreous space for monopolar delivery thereto.

FIG. 36 illustrates molecules disposed within the subretinal bleb and two electrode bodies positioned in the eye to deliver the energy from one to another in a bipolar manner.

FIG. 37 illustrates molecules disposed within the vitreous space and two electrode bodies positioned in the eye to deliver the energy from one to another in a bipolar manner.

FIG. 38 illustrates molecules disposed within the subretinal bleb and vitreous space and two electrode bodies positioned in the eye to deliver the energy from one to another in a bipolar manner.

FIG. 39 illustrates bipolar energy delivery to the eye wherein one electrode body is disposed in the vitreous space and one electrode body is disposed in a retrobulbar space, while molecules are disposed within a subretinal bleb.

FIG. 40 illustrates bipolar energy delivery to the eye wherein one electrode body is disposed in a subretinal bleb and one electrode body is disposed in a retrobulbar space, while molecules are disposed within the subretinal bleb.

FIG. 41 illustrates bipolar energy delivery to the eye wherein one electrode body is disposed in a subretinal bleb and one electrode body is disposed in a retrobulbar space, while molecules are disposed within the vitreous space.

FIG. 42 illustrates bipolar energy delivery to the eye wherein one electrode body is disposed in a subretinal bleb and one electrode body is disposed in a retrobulbar space, while molecules are disposed within the subretinal bleb and within the vitreous space.

FIG. 43 illustrates an embodiment wherein the molecules are delivered to the vitreous space and a first electrode body is positioned within the vitreous space while a second electrode body is positioned against the cornea.

FIGS. 44A-44B illustrate embodiments of an energy delivery device configured to deliver a solution, such as containing molecules, to target tissue.

FIG. 45 illustrates an embodiment of an energy delivery device showing a close up of its distal end.

DETAILED DESCRIPTION

Devices, systems and methods are provided for delivering molecules, particularly small molecules and/or macromolecules, to cells within the body, particularly to target cells which directly therapeutically benefit from the functionality of the molecules. Such delivery is considered transfer or biotransfer. Example molecules include DNA plasmids, RNAs (e.g. messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA), oligonucleotides, antisense oligonucleotides (ASO), proteins and/or materials which invoke genetic or epigenetic changes in the cellular behavior, to name a few. In such instances, the molecules are driven into the target cells with the use of pulsed electric fields (PEFs) which deliver the molecules through the cell wall of the target cells so that the desired genes are able to carry out the desired effect within the cells. Thus, the molecules are to be delivered to the desired location within the body at a desired time and in a desired concentration relative to the delivery of the pulsed electric fields for optimal outcomes, as will be described herein. The molecules may be delivered to the body by a variety of methods, such as systemically, regionally and/or by direct injection to the area of the target cells. The pulsed electric field energy is delivered to the target cells in vivo and in situ which drives the molecules into the cells. Thus, the molecules are passed into the cells without the use of viruses or ex vivo methods, such as in vitro electroporation.

These devices, systems and methods are superior to delivery by other methods, such as by adeno associated virus (AAV), a known virus-based gene therapy. AAV has a limited payload capacity; AAV can only accommodate genes<4.4 kb and has limited space for genetic control elements (e.g. promoters, etc). There are more than 300 disease genes that are too large for AAV-based gene therapies. These include genes leading to many relatively common diseases, such as Stargardt disease, Usher 1B and 1D, Leber congenital amaurosis-10 (LCA10), and cystic fibrosis. In contrast, the non-viral delivery described herein can accommodate genes>10 kb and is not limited in space. Thus, many of the genes precluded from use with AAV delivery can be delivered by the devices, systems and methods described herein. In addition, many patients have pre-existing anti-AAV antibodies, precluding their treatment, and immune responses against viral proteins or sequences prevents re-treatment. Such immunologic considerations are not relevant to the non-viral delivery methods described herein and pulsed electric fields are typically well tolerated by patients. Further, AAV gene therapy can only be targeted to tissues and cell types amenable to existing AAV serotypes. Such limitations are also not applicable to the non-viral delivery methods described herein since pulsed electric field delivery is applicable to all cell types and tissues.

These devices, systems and methods are also superior to delivery of cells to the body that have been previously transfected by in vitro laboratory techniques. As mentioned previously, transfecting cells in culture requires several additional steps when translating such transfection to a therapy for patients. These steps add substantial burden, cost, invasiveness, time, and risk to delivering a gene therapy.

Although the drawbacks of viral delivery and apheresis-based delivery are avoided with the devices, systems and methods described herein, direct transfer of molecules to cells within the body with the use of PEFs faces a number of challenges. To begin, such transfer involves delivery of a variety of devices and components to a target location in the body that naturally has anatomical and physiological constraints. Devices involved in the delivery of the molecules for transfer and/or the PEF energy are specially designed for accessing target tissue within the body, such as endoscopically, and delivering the desired components to the target cells.

In addition, the body presents a highly varied set of target tissues and local environments that are ever changing in the face of physiological reactions, conditions and processes. Protocols involved in the delivery of the molecules and the PEF energy are tailored to maximize transfer in various settings and in the face of changing environments. In some instances, changes in environment are induced to benefit the protocol and therefore outcome. This is particularly the case when delivering and concentrating the molecules within a desired target location within the body for transfer to the target cells.

Other challenges include delivering the PEF energy to the target cells within the body in a manner which causes transfer of the molecule but minimizes any potential deleterious effects to the target cells themselves and/or to any surrounding cells. In some instances, PEF energy can be used to ablate tissue rather than transfer molecules. Ablation involves cell death which is substantially contrary to the desired outcome of gene therapy and other therapies that rely on cell survival at least long enough to produce and invoke the therapeutic effect of the transferred molecules. In some instances, PEFs are able to kill cells via an array of non-thermal and thermal mechanisms, based on the PEF waveform and protocol employed. This is particularly relevant when high voltages are utilized. Therefore, to generate more transfer, simply increasing the voltage may be detrimental, leading to undesired cell death. Thus, numerous variables are carefully optimized and balanced in order to achieve transfer without lethality.

In some instances, device design is optimized to control for the unique situation of transfer within the body. Energy emanating from an electrode on a device, such as in a monopolar fashion with the use of a remote return electrode, may result in cells closest to the electrode receiving high energy levels with an exponential drop off with distance from the electrode. Thus, energy bands may result in cells closest to an electrode potentially dying while cells further away received transferred cells and cells even further away are not affected. These bands may all exist within a few millimeters from an electrode. Increasing the voltage may exacerbate such cell death making large-volume and/or high total-cell count transfer difficult. These challenges are unique to delivery in the body where the laboratory techniques of plate electrodes at controlled distances are rarely applicable.

In addition, delivery in the body involves mindfulness of the entire body environment and potential reactions to the delivery procedure. In particular, undesired muscle contraction is a potential outcome, particularly when using conventional methods of transfection designed for in vitro laboratory techniques. Such techniques involve delivery of long duration (e.g. 100 s of μs to 100 s of ms) waveforms as part of the transfection procedure. In vivo, this activates the motor neurons and skeletal muscle which can cause patient injury (e.g. falling off a table, movement of a device within the patient's body, etc.), cause pain (e.g. requiring local or general anesthesia) or can affect therapy outcome (e.g via electrode migration over the course of a treatment). Neuromuscular paralytics can help reduce this risk, but this involves general anesthesia and is often insufficient in sufficiently mitigating the contraction.

The devices, systems and method described herein overcome these unique challenges, bringing PEF transfer in the body to the clinically relevant, translatable, forefront of potential therapy techniques.

The devices, systems and methods deliver energy to the cells with an energy delivery system. Generally, the energy delivery systems include a specialized energy delivery device, a waveform generator and at least one distinct energy delivery algorithm. Additional accessories and equipment may be utilized. For example, in some embodiments, the energy delivery device is delivered through an endoscope, typically specific to the anatomical location to which it is being used, such as gastroscopes (upper GI endoscopy, which includes the stomach, esophagus, and small intestine (duodenum)), colonoscopes (large intestine), bronchoscopes (lungs), laryngoscopes (larynx), cystoscopes (urinary tract), duodenoscopes (small intestine), enteroscopes (digestive system), ureteroscopes (ureter), hysteroscopes (cervix, uterus), etc. It may be appreciated that in other embodiments, the energy deliver device is deliverable through a catheter, sheath, introducer, needle or other delivery system.

Endoluminal access allows treatment of target tissue from within various lumens in the body. Lumens are the spaces inside of tubular-shaped or hollow structures within the body and include passageways, canals, ducts and cavities to name a few. Example luminal structures include blood vessels, esophagus, stomach, small and large intestines, colon, bladder, urethra, urinary collecting ducts, uterus, vagina, fallopian tubes, ureters, kidneys, renal tubules, spinal canal, spinal cord, and others throughout the body, as well as structures within and including such organs as the lung, heart and kidneys, to name a few. In some embodiments, the target tissue is accessed via the nearby luminal structure. In some instances, an energy delivery device is advanced through various luminal structures or branches of a luminal system to reach the target tissue location. For example, when accessing a target tissue site via a blood vessel, the energy delivery device may be inserted remotely and advanced through various branches of the vasculature to reach the target site. Likewise, if the luminal structure originates in a natural orifice, such as the nose, mouth, urethra or rectum, entry may occur through the natural orifice and the energy delivery device is then advanced through the branches of the luminal system to reach the target tissue location. Alternatively, a luminal structure may be entered near the target tissue via cut-down or other methods. This may be the case when accessing luminal structures that are not part of a large system or that are difficult to access otherwise.

It may be appreciated that a variety of anatomical locations may be treated endoluminally with the systems and methods described herein. Examples include luminal structures themselves, soft tissues throughout the body located near luminal structures and solid organs accessible from luminal structures, including but not limited to liver, pancreas, gall bladder, kidney, prostate, ovary, lymph nodes and lymphatic drainage ducts, underlying musculature, bony tissue, brain, eyes, thyroid, etc. It may also be appreciated that a variety of tissue locations can be accessed percutaneously or by other methods.

Target tissue cells may be treated in any location throughout the body, including cells of the digestive system (e.g. mouth, glands, esophagus, stomach, duodenum, jejunum, ileum, intestines, colon, rectum, liver, gall bladder, pancreas, anal canal, etc.), cells of the respiratory system (e.g. nasal cavity, pharynx larynx, trachea, bronchi, lungs, etc.), cells of the urinary system (e.g. kidneys, ureter, bladder, urethra, etc.), cells of the reproductive system (e.g. reproductive organs, ovaries, fallopian tubes, uterus, cervix, vagina, testes, epididymis, vas deferens, seminal vesicles, prostate, glands, penis, scrotum, etc.), cells of the endocrine system (e.g. pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland), cells of the circulatory system (e.g heart, arteries, veins, etc.), cells of the lymphatic system (e.g. lymph node, bone marrow, thymus, spleen, etc.), cells of the nervous system (e.g. brain, spinal cord, nerves, ganglia, etc.), cells of the eye (e.g. retina, macula, Layer of Rods and Cones, retinal pigment epithelium optic nerve, choroid, sclera, etc.), cells of the muscular system (e.g. myocytes, etc.), and cells of the skin (e.g. epidermis, dermis, hypodermis, etc.), to name a few.

The energy delivery device delivers energy provided by the waveform generator according to the at least one distinct energy delivery algorithm. It may be appreciated that, in some embodiments, the energy delivery device also delivers the molecules. However, in other embodiments, the molecules are delivered by a separate device, such as by IV, catheter, or needle injection. Optionally, the molecules may be delivered both by the energy delivery device and by a separate device. Example embodiments of specialized energy delivery devices are provided herein primarily focused on monopolar energy delivery, however, it may be appreciated that bipolar or multi-polar arrangements may be used.

FIG. 2 illustrates an embodiment of an energy delivery system 100 comprising a specialized energy delivery device 102, a return electrode 115, and a waveform generator 104. In this embodiment, the target tissue is located within a liver L of a patient P. In this embodiment, the energy delivery device 102 comprises a flexible elongate shaft having a distal end capable of being advanced endoluminally to the target tissue within the liver L. As shown, the distal end of the delivery device 102 is advanced through the mouth M, down the esophagus E, into the stomach S wherein it passes through the stomach wall into the liver L. In some embodiments, the distal end has a distal tip 103 configured to penetrate the stomach wall and/or the liver L. In other embodiments, a passageway is formed through the stomach wall with the use of a separate instrument which is then removed so that an energy delivery device 102 having an atraumatic tip is able to be passed through the passageway.

In this embodiment, molecules 110 are delivered systemically, intravenously with the use of an IV bag 112. This typically disperses the molecules 110 throughout the body of the patient P, including to the target tissue within the liver L. It may be appreciated that in other embodiments, the molecules 110 are delivered regionally. In such embodiments, the molecules 110 may be delivered to the vasculature, upstream of the arterial system that leads to the targeted organ or tissue area. The molecules 110 then travel through the downstream arterial circulation into the targeted region. If a bolus injection of the molecules 110 is provided, a sudden rush of molecules 110 will enter into the targeted tissue. However, if the molecules 110 are delivered over time, such as with the use of an infusion pump, a steady, sustained level of molecules 110 may be achieved in the targeted tissue. It may be appreciated that in other embodiments, the molecules 110 are delivered by direct injection to the targeted tissue. In such embodiments, the injection device is inserted in or near the targeted tissue, such as within the parenchymal tissue of the targeted organ region, and a solution containing the affecter genetic material is injected. The solution of molecules may be permitted a period of time for its distribution through the parenchyma and interstitial spaces to reach an area or volume targeted for transfer. It may be appreciated that any combination of systemic, regional and local delivery may alternatively be used.

Pulsed electric field energy is delivered to the target tissue through the distal end of the delivery device 102. The proximal end of the delivery device 102 is electrically connected with the waveform generator 104. In some embodiments, the generator 104 is also connected with an external cardiac monitor (not shown) to allow coordinated delivery of energy with the cardiac signal sensed from the patient P.

In this embodiment, the energy delivery device 102 is designed to be monopolar, wherein the distal end of the delivery device 102 has as a delivery electrode and the return electrode 115 is positioned upon the skin outside the body, typically on the thigh (as shown), lower back or back.

Pulsed electric fields (PEFs) are provided by the generator 104 and delivered to the tissue through an energy delivery body 108 placed on, in, or near the targeted tissue area. It may be appreciated that in some embodiments, the energy delivery body 108 is positioned in contact with a conductive substance which is likewise in contact with the targeted tissue. Such solutions may include isotonic or hypertonic solutions. Electric pulses are then delivered through the energy delivery body 108 in the vicinity of the target tissue. These electric pulses are provided by at least one energy delivery algorithm 152. The algorithm 152 specifies various parameters of the signal which contribute to the overall shape of the waveform, such as energy amplitude (e.g. voltage) and duration of applied energy, which is comprised of the number of pulses, the pulse widths and the delay between pulses, to name a few. In some embodiments, one or more of the energy delivery bodies are small and tend to dissipate large amount of energy around the electrode. Therefore, an optimal delivery of energy is desired. In some embodiments, a large DC-link capacitance with half transistor bridges is utilized to deliver efficient delivery pulses in such instances. In some instances, this is preferred in relation to pulse voltages delivered by power amplifiers (limited bandwidth) or exponential decay generators. In some embodiments, a feedback loop based on sensor information and an auto-shutoff specification, and/or the like, may be included.

As will be described in later sections, in some embodiments, biphasic pulses may be used. In such embodiments, additional parameters may include switch time between polarities in biphasic pulses and dead time between biphasic cycles. Biphasic waveforms may be used to reduce muscle stimulation in patients. This is particularly important in the application where slight movement of the energy delivery body can result a non-effective therapy or detrimental consequences. Biphasic waveforms involve rapid change of phases/polarities of the signal to minimize nerve activation during transition between polarity. Multiple fast switching elements (e.g. MOSFET, IGBT Transistors) are desired and are employed and configured in, for example, H-bridge structure or full bridge.

Referring back to FIG. 1 , in this embodiment the generator 104 includes a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage/retrieval unit 156 (such as a memory and/or database), and an energy-storage sub-system 158 which generates and stores the energy to be delivered. In some embodiments, one or more capacitors are used for energy storage/delivery, however any other suitable energy storage element may be used. In addition, one or more communication ports may be included.

In some embodiments, the generator 104 includes three sub-systems: 1) a high-energy storage system, 2) a high-voltage, medium-frequency switching amplifier, and 3) the system controller, firmware, and user interface. The generator takes in alternating current (AC) mains to power multiple direct current (DC) power supplies. The generator's controller can cause the DC power supplies to charge a high-energy capacitor storage bank before energy delivery is initiated. In some embodiments, at the initiation of energy delivery, the generator's controller, high-energy storage banks and a bi-phasic pulse amplifier can operate simultaneously to create a high-voltage, medium frequency output.

It will be appreciated that a multitude of generator electrical architectures may be employed to execute the energy delivery algorithms. In particular, in some embodiments, advanced switching systems are used which are capable of directing the pulsed electric field circuit to the energy delivering electrodes separately from the same energy storage and high voltage delivery system. Further, generators employed in advanced energy delivery algorithms employing rapidly varying pulse parameters (e.g., voltage, frequency, etc.) or multiple energy delivery electrodes may utilize modular energy storage and/or high voltage systems, facilitating highly customizable waveform and geographical pulse delivery paradigms. It should further be appreciated that the electrical architecture described herein above is for example only, and systems delivering pulsed electric fields may or may not include additional switching amplifier components.

The user interface 150 can include a touch screen and/or more traditional buttons to allow for the operator to enter patient data, select a treatment algorithm (e.g., energy delivery algorithm 152), initiate energy delivery, view records stored on the storage/retrieval unit 156, and/or otherwise communicate with the generator 104.

In some embodiments, the user interface 150 is configured to receive operator-defined inputs. The operator-defined inputs can include a duration of energy delivery, one or more other timing aspects of the energy delivery pulse, power, and/or mode of operation, or a combination thereof. Example modes of operation can include (but are not limited to): system initiation and self-test, operator input, algorithm selection, pre-treatment system status and feedback, energy delivery, post energy delivery display or feedback, treatment data review and/or download, software update, or any combination or subcombination thereof.

In some embodiments, the processor 154, among other activities, modifies and/or switches between the energy-delivery algorithms, monitors the energy delivery and any sensor data, and reacts to monitored data via a feedback loop. In some embodiments, the processor 154 is configured to execute one or more algorithms for running a feedback control loop based on one or more measured system parameters (e.g., current), one or more measured tissue parameters (e.g., impedance), and/or a combination thereof.

The data storage/retrieval unit 156 stores data, such as related to the treatments delivered, and can optionally be downloaded by connecting a device (e.g., a laptop or thumb drive) to a communication port. In some embodiments, the device has local software used to direct the download of information, such as, for example, instructions stored on the data storage/retrieval unit 156 and executable by the processor 154. In some embodiments, the user interface 150 allows for the operator to select to download data to a device and/or system such as, but not limited to, a computer device, a tablet, a mobile device, a server, a workstation, a cloud computing apparatus/system, and/or the like. The communication ports, which can permit wired and/or wireless connectivity, can allow for data download, as just described but also for data upload such as uploading a custom algorithm or providing a software update.

As described herein, a variety of energy delivery algorithms 152 are programmable, or can be pre-programmed, into the generator 104, such as stored in memory or data storage/retrieval unit 156. Alternatively, energy delivery algorithms can be added into the data storage/retrieval unit to be executed by processor 154. Each of these algorithms 152 may be executed by the processor 154.

In some embodiments, the energy delivery device 102 includes one or more sensors that can be used to determine temperature, impedance, resistance, capacitance, conductivity, pH, optical properties (coherence, echogenicity, fluorescence), electrical or light permittivity, and/or conductance, to name a few. In some embodiments, one or more of the electrodes act as the one or more sensors. In other embodiments, the one or more sensors are separate from the electrodes. Sensor data can be used to plan the therapy, monitor the therapy and/or provide direct feedback via the processor 154, which can then alter the energy-delivery algorithm 152. For example, impedance measurements can be used to determine not only the initial dose to be applied but can also be used to determine the need for further energy delivery, or not.

It may be appreciated that in some embodiments the system 100 includes an automated treatment delivery algorithm that dynamically responds and adjusts and/or terminates delivery in response to inputs such as temperature, impedance at various voltages or AC frequencies, time duration or other timing aspects of the energy delivery pulse, treatment power and/or system status.

Device Embodiments

The energy may be delivered by a variety of energy delivery devices 102. Typically, the energy delivery device 102 comprises a flexible elongate shaft having a distal end, capable of being advanced to the target tissue with the body, and at least one energy delivery body 108 disposed near the distal end. The energy delivery body 108 comprises one or more electrodes that delivers the PEF energy to the target tissue.

As mentioned previously, in some embodiments the energy delivery device 102 delivers the PEF energy and the molecules 110 are delivered by a separate device, such as by IV, catheter, or needle injection. FIG. 3A illustrates direct injection of molecules 110 to a target tissue through a needle 500. The target tissue is illustrated as cells C (not to scale). The needle 500 is inserted in or near the target tissue so that the injected molecules 110 are able to bathe the target tissue. In this embodiment, the needle 500 is then removed and the molecules 110 dwell for biodistribution. Referring to FIG. 3B, the distal end of the delivery device 102 is then inserted into the target tissue so that the energy delivery body 108 is desirably positioned within or near the target tissue. In this embodiment, the energy delivery body 108 is comprised of a single electrode. PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

In some embodiments, the molecules 110 and the energy are delivered by the energy delivery device 102. FIGS. 4A-4B illustrate an energy delivery device 102 having an energy delivery body 108 having a needle shape. The tip of the needle shape is able to penetrate similarly to a needle and deliver molecules 110 through its internal lumen. In addition, the energy delivery body 108 is electrically insulated with an insulation layer 504 except for the tip of the needle shape which acts as an electrode. FIG. 4A illustrates direct injection of molecules 110 to a target tissue through the energy delivery body 108. Again, the target tissue is illustrated as cells C (not to scale). The tip is inserted in or near the target tissue so that the injected molecules 110 are able to bathe the target tissue and optionally dwells for biodistribution. Referring to FIG. 4B, PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

FIG. 5 illustrates molecules 110 delivered regionally while energy is delivered locally (and optionally molecules 110 are additionally delivered locally). Here, molecules 110 are delivered by a separate device, such as a catheter 501 positioned within the vasculature V that feeds the target tissue area. Thus, molecules 110 are delivered regionally to the target tissue area. The energy delivery device 102 is inserted into the target tissue area from a different approach. Here, the energy delivery device 102 comprises an energy delivery body 108 having a needle shape. The tip of the needle shape is able to penetrate similarly to a needle. In some embodiments, molecules 110 are able to be delivered through its internal lumen. In this embodiment, the energy delivery body 108 is electrically insulated with an insulation layer 504 except for the tip of the needle shape which acts as an electrode. The tip is inserted in or near the target tissue and PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the target cells.

FIG. 6 illustrates an energy delivery device 102 comprising a shaft 106 having an energy delivery body 108 near its distal end, wherein the energy delivery body 108 comprises a plurality of tines 600. Typically, the tines 600 have a pointed shape so as to penetrate tissue. Likewise, the tines 600 typically extend laterally outward from the shaft 106, and in some embodiments the tines 600 are deployed circumferentially around the shaft 106. It may be appreciated that in some embodiments, the tines 600 are deployed from a side of the shaft 106, such as aligned in a row. In some embodiments, the tines 600 extend the same distance from the shaft 106 and in other embodiments the tines 600 extend a varied distance. It may be appreciated that in some embodiments, the extension of at least some of the tines 600 from the shaft 106 is adjustable.

Typically, each tine 600 delivers molecules 110 and/or energy therefrom. In some embodiments, molecules 110 are delivered from the tip 601 of the tine 600 and in other embodiments molecules 110 are delivered from delivery ports 602 along the tine 600. In some embodiments, the tines 600 are energizable together (e.g. so as to act as a single electrode) or at least some of the tines 600 are individually energizable (e.g. so as to act in bipolar pairs or so as to act as selectable single electrodes including acting in groups). In some embodiments, one or more tines 600 deliver different energy (e.g. generated from different energy delivery algorithms 152) and/or different types of molecules 110.

In this embodiment, the shaft 106 has three sections, a first section 106 a, a second section 106 b and a third section 106 c. As illustrated in FIG. 6 , the first section 106 a is distal to the second section 106 b which is distal to the third section 106 c. Each section 106 a, 106 b, 106 c may be insulated or non-insulated so as to create a variety of different electrode combinations. This may allow various electric field shapes and/or direct the electric field in desired directions. It may also be appreciated that in some embodiments, at least a portion of at least one tine 600 is insulated so as to direct the energy emanating therefrom. Overall, the tines 600 are often able to deliver molecules 110 and/or energy to a larger volume of target tissue with a single placement of the energy delivery device 102 than with a device 102 having an energy delivery device 108 comprising a single needle.

In some embodiments, the first section 106 a acts as an energy delivery body 108 and one or more tines 600 act as energy delivery bodies 108. Each of the different energy delivery bodies 108 may deliver the same or different types of energy; likewise, the energy delivery bodies 108 may act in groups. In some embodiments, the tines 600 extend past the first section 106 a. In some embodiments, the tines 600 extend the same distance (relative to the first section 106 a) from the shaft 106 and in other embodiments the tines 600 extend a varied distance (relative to the first section 106 a). In some embodiments, the first section 106 a acts as an energy delivery body 108 and one or more tines 600 act as a conduit for delivery of molecules 110.

FIG. 7 illustrates an energy delivery device 102 comprising an energy delivery body 108 having a basket shape configured for treating target tissue endoluminally. Here the target tissue comprises cells C disposed near a wall W of a body lumen, particularly wrapping at least partially circumferentially around the body lumen. In this embodiment, the energy delivery body 108 is comprised of a plurality of wires or ribbons 120 forming a spiral-shaped basket serving as an electrode. In some embodiments, the energy delivery body 108 is self-expandable and delivered to a targeted area in a collapsed configuration. This collapsed configuration can be achieved, for example, by placing a sheath over the energy delivery body 108. Retraction of the sheath or advancement of the energy delivery body 108 from the sheath allows the energy delivery body 108 to self-expand. In other embodiments, the energy delivery device 102 includes a handle having an energy delivery body manipulation knob wherein movement of the knob causes expansion or retraction/collapse of the basket-shaped electrode. The basket-shaped electrode is expandable within a body lumen or passageway (naturally occurring or created within the body) so as to contact at least a portion of the wall W of the lumen. Molecules 110 are delivered from the energy delivery device 102, such as through a distal end port 510 and/or through various side ports 512 along a shaft 106 of the device 102, such as within the basket-shaped electrode, as illustrated in FIG. 7 . The molecules 110 are able to bathe the target tissue and optionally dwell for biodistribution. PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

FIG. 8 illustrates another embodiment of an energy delivery device 102 comprising an energy delivery body 108 having a shape configured for treating target tissue endoluminally. In this embodiment, the energy delivery body 108 comprises at least two protrusions 514, each protrusion extending radially outwardly so as to contact an inner luminal wall W. It may be appreciated that although a single protrusion may be present, typically two protrusions are present to apply substantially opposing forces to the walls the lumen. In the embodiment of FIG. 8 , three protrusions 514 are present. In some embodiments, each protrusion 514 is formed by a wire or ribbon which acts as an electrode and bends or bows radially outward from the longitudinal axis or shaft 106 of the delivery device 102. In this embodiment, the protrusions 514 together act as a single electrode. However, in other embodiments, one or more protrusions 514 are independently energizeable so as to act as multiple electrodes (e.g. as one or more bipolar pairs). The protrusions 514 may be comprised of a variety of suitable materials so as to act as an electrode, such as stainless steel, spring steel, or other alloys, and may be, for example, round wires or ribbon. In some embodiments, a portion of the protrusions 514 are insulated with a segment of insulation, such as a polymer (e.g., PET, polyether block amide, polyimide). For example, in some embodiments at least a portion of the proximal and distal ends of the energy delivery body 108 are insulated to direct the energy laterally, toward the walls W.

In some embodiments, the energy delivery body 108 of FIG. 8 is self-expandable and delivered to a targeted area in a collapsed configuration. The protrusions bow outwardly during expansion within a body lumen or passageway (naturally occurring or created within the body) so as to contact at least a portion of the wall W of the lumen. Molecules 110 is delivered from the energy delivery device 102, such as through a port 516 within the energy delivery body 108, as illustrated in FIG. 8 . The molecules 110 is able to bathe the target tissue and optionally dwell for biodistribution. PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

FIG. 9 illustrates another embodiment of an energy delivery device 102 comprising an energy delivery body 108 having a shape configured for treating target tissue endoluminally. In this embodiment, the energy delivery body 108 comprises an expandable member 518, such as an inflatable balloon, having an electrode 520 mounted thereon or incorporated therein. The energy delivery body 108 is delivered to a targeted area in a collapsed configuration. In this embodiment, the electrode 520 has the form of a pad having a relatively broad surface area and thin cross-section. The pad shape provides a broader surface area than other shapes, such as a wire shape. Each electrode 520 is connected with a conduction wire 522 which electrically connects the electrode 520 with the generator. In this embodiment, the three electrodes 520 are visible, however it may be appreciated that additional electrodes may be present around the expandable member 518. It may be appreciated that any number of electrodes 520 may be present, acting as a single electrode or acting independently or in combination. Placement of the electrodes 520 and/or selective energizing of the electrodes 520 may direct the energy toward particular target locations. In some embodiments, the electrodes 520 are comprised of flexible circuit pads or other materials attached to the expandable member 518 or formed into the expandable member 518. In some embodiments, the electrodes 520 are distributed radially around the circumference of the expandable member 518 and/or distributed longitudinally along the length of the expandable member 518. Such designs may facilitate improved deployment and retraction qualities, easing user operation and compatibility with introducer lumens.

Upon expansion of the expandable member, one or more of the electrodes 520 are positioned so as to contact at least a portion of the wall W of the lumen. Molecules 110 are delivered from the energy delivery device 102, such as through a distal end port 510, as illustrated in FIG. 9 . The molecules 110 are able to bathe the target tissue and optionally dwell for biodistribution. PEF energy is then delivered to the target tissue from the energy delivery body 108 as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

FIG. 10 illustrates another embodiment of an energy delivery device 102. Here the energy delivery body 108 has a finger-tip shape configured to contact an inner lumen wall W. In this embodiment, the energy delivery device 102 has an elongate shaft 106 and finger-tip electrode 530 disposed at its distal tip. The finger-tip electrode 530 is positionable against the portion of the lumen wall W near the target tissue cells C. The molecules 110 may be delivered by any suitable method, such as systemically, regionally or locally, such as by injection through a separate device or through the energy delivery device 102. FIG. 10 illustrates delivery of molecules 110 through the finger tip electrode 530. Energizing the finger-tip electrode 530 directs the PEF energy toward the cells C as indicated by wavy lines 502. The PEF energy transfers the molecules 110 into the cells C.

It may be appreciated that in some embodiments, PEF energy is delivered to a conductive fluid (e.g. blood, saline, etc.) in contact with the target tissue. Thus, the energy is able to pass through the conductive fluid to the target tissue for transfer. In other embodiments, delivery of energy to the conductive fluid promotes transfer of genetic material into the cells of the fluid itself, such as transfer into leukocytes in blood.

Molecules

As mentioned previously, the devices, systems and methods are provided for delivering molecules 110, particularly small molecules and/or macromolecules, to cells within the body, such as to target cells which directly therapeutically benefit from the functionality of the molecules. Such therapeutic benefit may be in the treatment of a variety of disorders.

In some embodiments, the disorder comprises a coagulation disorder, such as hemophilia (e.g., hemophilia A or hemophilia B), von Willebrand's disease, factor XI deficiency, a fibrinogen disorder, or a vitamin K deficiency. The coagulation disorder may be characterized by a mutation in a gene encoding for fibrinogen, prothrombin, factor V, factor VII, factor VIII, factor X, factor XI, factor XIII, or an enzyme involved in posttranslational modifications thereof, or an enzyme involved in vitamin K metabolism. In some embodiments, the coagulation disorder is characterized by a mutation in FGA, FGB, FGG, F2, F5, F7, F10, F11, F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

In some embodiments, the disorder comprises a neurological disorder, e.g., a neurodegenerative disease. In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, or multiple sclerosis. In some embodiments, the neurodegenerative disease comprises an autoimmune disease of the central nervous system (CNS), such as multiple sclerosis, encephalomyelitis, a paraneoplastic syndrome, autoimmune inner ear disease, or opsoclonus myoclonus syndrome. The neurological disorder may be a cerebral infarction, spinal cord injury, central nervous system disorder, a neuropsychiatric disorder, or a channelopathy (e.g., epilepsy or migraine). The neurological disorder may be an anxiety disorder, a mood disorder, a childhood disorder, a cognitive disorder, schizophrenia, a substance related disorders, or an eating disorder. In some embodiments, the neurological disorder is a symptom of a cerebral infarction, stroke, traumatic brain injury, or spinal cord injury.

In some embodiments, the disorder comprises a lysosomal storage disorder, such as Tay-Sachs disease, Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, or mucopolysaccharidosis (MPS).

In some embodiments, the disorder comprises a cardiovascular disorder, such as a degenerative heart disease, a coronary artery disease, an ischemia, angina pectoris, an acute coronary syndrome, a peripheral vascular disease, a peripheral arterial disease, a cerebrovascular disease, or atherosclerosis. The cardiovascular disorder may be a degenerative heart disease selected from the group consisting of an ischemic cardiomyopathy, a conduction disease, and a congenital defect.

In some embodiments, the disorder comprises an immune disorder, e.g., an autoimmune disorder. The autoimmune disorder may be type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, encephalomyelitis, a paraneoplastic syndrome, autoimmune inner ear disease, or opsoclonus myoclonus syndrome, autoimmune hepatitis, uveitis, autoimmune retinopathy, neuromyelitis optica, psoriatic arthritis, psoriasis, myasthenia gravis, chronic Lyme disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, peripheral neuropathy, fibromyalgia, Hashimoto's thyroiditis, ulcerative colitis, or Kawasaki disease.

In some embodiments, the disorder comprises a liver disease, such as hepatitis, Alagille syndrome, biliary atresia, liver cancer, cirrhosis, a cystic disease, Caroli's syndrome, congenital hepatic fibrosis, fatty liver, galactosemia, primary sclerosing cholangitis, tyrosinemia, glycogen storage disease, Wilson's disease, or an endocrine deficiency. The liver disease may be a liver cancer such as a hepatocellular hyperplasia, a hepatocellular adenoma, a focal nodular hyperplasia, or a hepatocellular carcinoma.

In some embodiments, the disorder comprises a cancer, such as a blood cancer (e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia, chromic myelogenous leukemia, Hodgkin's disease, multiple myeloma, and non-Hodgkin's lymphoma) or a solid tissue cancer (e.g., liver cancer, kidney cancer, a breast cancer, a gastric cancer, an esophageal cancer, a stomach cancer, an intestinal cancer, a colorectal cancer, a bladder cancer, a head and neck cancer, a skin cancer, or a brain cancer).

In some embodiments, the disorder comprises a recessively inherited disorder. In some embodiments, the disorder is a Mendelian-inherited disorder.

In some embodiments, the disorder comprises an ocular disorder that is a retinal dystrophy (e.g., a Mendelian-heritable retinal dystrophy). The retinal dystrophy may be comprised of leber's congenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, stickler syndrome, microcephaly and choriorretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.

In some embodiments, the molecules 110 delivered by the devices, systems and methods described herein include synthetic DNA vectors, such as those described in Publication No. WO2019178500 filed on Mar. 15, 2019, entitled “Synthetic DNA Vectors and Methods of Use,” incorporated in its entirety herein for all purposes. Such synthetic DNA vectors include non-viral DNA vectors, such as those that provide long-term transduction of quiescent cells (e.g., post-mitotic cells) in a manner similar to AAV vectors. In some embodiments, such non-viral DNA vectors are development by an in vitro (e.g., cell-free) system to synthetically produce circular AAV-like DNA vectors (e.g., DNA vectors containing a terminal repeat sequence, such as a DD element) by isothermal rolling-circle amplification and ligation-mediated circularization (as opposed to bacterial expression and site-specific recombination, for example). Such development allows for improved scalability and manufacturing efficiency in production of circular AAV-like DNA vectors. Moreover, the vectors produced by these methods are designed to overcome many of the problems associated with plasmid-DNA vectors, e.g., problems discussed in Lu et al., Mol. Ther. 2017, 25(5): 1187-98, which is incorporated herein by reference in its entirety. For example, by eliminating or reducing the presence of CpG islands and/or bacterial plasmid DNA sequences such as RNAPII arrest sites, transcriptional silencing can be reduced or eliminated, resulting in increased persistence of the heterologous gene. Further, by eliminating the presence of immunogenic components (e.g., bacterial endotoxin, DNA, or RNA, or bacterial signatures, such as CpG motifs), the risk of stimulating the host immune system is reduced. Such benefits are especially advantageous in the treatment of certain disorders, such as retinal dystrophies (e.g., Mendelian-heritable retinal dystrophies).

Thus, such vectors include synthetic DNA vectors that: (i) are substantially devoid of bacterial plasmid DNA sequences (e.g., RNAPII arrest sites, origins of replication, and/or resistance genes) and other bacterial signatures (e.g., immunogenic CpG motifs); and/or (ii) can be synthesized and amplified entirely in a test tube (e.g., replication in bacteria is unnecessary, e.g., bacterial origins of replication and bacterial resistance genes are unnecessary). In some embodiments, the vectors contain a double-D (DD) element characteristic of AAV vectors. This allows a target cell to be transduced with a DNA vector having a heterologous gene that behaves like AAV viral DNA (e.g., having low transcriptional silencing and enhanced persistence), without needing the virus itself.

In some embodiments, the molecules 110 include nucleic acid-based molecules, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), oligonucleotides, antisense oligonucleotide (ASO), microRNA (miRNA), decoy DNA, ribozyme, morpholino and plasmid. RNA interference using small inhibitory RNA (siRNA) can be used to downregulate mRNA levels by cellular nucleases that become activated when a sequence homology between the siRNA and a respective mRNA molecule is detected. Therefore, in some embodiments, siRNA is used to silence genes involved in the pathogenesis of various diseases associated with a known genetic background. In some embodiments, the molecules 110 comprise patisiran, an siRNA-based drug FDA approved for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis. In order for the siRNA to function, the siRNA must be inside the target cell of interest. This means the siRNA must be transported to the tissue in the body where the target cells reside and then it must cross through the cell's membrane. These requirements are generally referred to as “delivery” of the siRNA to the desired location. Delivery has proved difficult in conventional delivery methods because siRNAs are negatively charged molecules that do not naturally cross through a cell's outer membrane. The devices, systems and methods described herein overcome these delivery difficulties, delivering the siRNA into the target cells.

In some embodiments, the molecules 110 include microRNAs (miRNAs). miRNAs are a class of small noncoding RNAs of ˜22 nt in length which are involved in the regulation of gene expression at the posttranscriptional level by degrading their target mRNAs and/or inhibiting their translation.

In some embodiments, the molecules 110 include antisense oligonucleotides (ASO). ASOs are synthetic DNA oligomers that hybridize to a target RNA in a sequence-specific manner. In some embodiments, ASOs are delivered to inhibit gene expression, modulate splicing of a precursor messenger RNA, or inactivate microRNAs. In order to stabilize ASO against nucleolytic degradation, chemically modified nucleotides such as phosphorothioates, 2′-O-methyl RNA, or locked nucleic acids may be used because they confer nuclease resistance. In some embodiments, ASOs are delivered with optimization of enhanced delivery, specificity, affinity, and nuclease resistance with reduced toxicity.

Example ASOs include (1) fomivirsen, such as for treatment of CMV retinitis in AIDS patients, (2) mipomersen, such as for treatment of familial hypercholesterolemia, (3) defibrotide, such as for treatment of veno-occlusive disease in the liver, (4) eteplirsen, such as for the treatment of Duchenne muscular dystrophy, (5) pegaptanib, such as for the treatment of neovascular age-related macular degeneration, and (6) nusinersen, such as for the management of spinal muscular atrophy.

In some embodiments, the molecules 110 include oligomer molecules, such as phosphorodiamidate Morpholino oligomer (PMO), also known as Morpholino, a type of oligomer molecule used to modify gene expression knocking down gene function. Usually 25 bases in length, Morpholinos bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing. A Morpholino oligo specifically binds to its selected DNA or RNA target site to block access of cell components to that site. This property can be exploited to block translation, block splicing, block microRNAs (miRNAs) or their targets, and block ribozyme activity. Its molecular structure contains DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Because the uncharged backbone of the Morpholino oligo is not recognized by enzymes, it is completely stable to nucleases. In some embodiments, the Morpholino-based drug, eteplirsen, is delivered which may be used in the treatment of some mutations causing Duchenne muscular dystrophy (DMD). In other embodiments, the Morpholino-based drug, golodirsen, is delivered for DMD treatment.

In some embodiments, the molecules 110 include ribozymes (ribonucleic acid enzymes) which are naturally occurring RNA molecules that catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. In some embodiments, the molecules 110 comprise synthetic ribozymes, such as designed to inhibit the production of proteins through the specific cleavage of the disease-causing mRNA. Another application of ribozyme therapy includes the inhibition of RNA-based viruses such as HIV, hepatitis C virus, SARS coronavirus (SARS-CoV), Adenovirus and influenza A and B virus.

In some embodiments the molecules 110 comprise a ribonucleoprotein (RNP). RNP is a complex formed between RNA and RNA-binding proteins. For instance, purified Cas9 Protein can be combined with guide RNA to form an RNP complex to be delivered to cells for rapid and highly efficient genome editing. RNPs remain in the cell for a short time and the dose is minimal, leading to lower toxicity and reduced editing at off-target sites compared to other methods. RNP complex are also DNA-free lacking therefore insertional mutagenesis risks.

In some embodiments, the molecules 110 delivered by the devices, systems and methods described herein include Clustered Regularly Interspaced Short Palindromic Repeats Repetitive (CRISPR) DNA sequences, called CRISPR. These DNA sequences were originally observed in bacteria with “spacer” DNA sequences in between the repeats that exactly match viral sequences. It was subsequently discovered that bacteria transcribe these DNA elements to RNA upon viral infection. The RNA guides a nuclease (a protein that cleaves DNA) to the viral DNA to cut it, providing protection against the virus. The nucleases are named “Cas,” for “CRISPR-associated.”

In 2012, researchers demonstrated that RNAs could be constructed to guide a Cas nuclease (Cas9 was the first used) to any DNA sequence. The so-called guide RNA can also be made so that it will be specific to only that one sequence, improving the chances that the DNA will be cut at that site and nowhere else in the genome. Further testing revealed that the system works quite well in all types of cells, including human cells.

With CRISPR/Cas, a targeted gene is able to be disrupted, or, if a DNA template is added to the mix, a new sequence is able to be inserted at a precise spot desired. The method has been used to develop animal models with specific genomic changes. And for human diseases with a known mutation, such as cystic fibrosis, it is theoretically possible to insert DNA that corrects the mutation. However, it has been difficult to deliver the CRISPR/Cas material to mature cells in large numbers using conventional methods such as viral vectors. However, the devices, systems and methods described herein overcome these difficulties allowing molecules 110 comprising CRISPR/Cas material to be delivered to cells.

In some embodiments, the molecules 110 comprise recombinant protein. With the use of recombinant DNA technology, such therapeutic proteins have been developed to treat a wide variety of disease, including cancers, autoimmunity/inflammation, exposure to infectious agents, and genetic disorders.

In some embodiments, the molecules 110 comprise a proteolysis targeting chimera (PROTAC). PROTAC is a small molecule capable of removing specific unwanted proteins. PROTACs are comprised two covalently linked protein-binding molecules: one capable of engaging an E3 ubiquitin ligase, and another that binds to a target protein meant for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. At present, PROTACs have been successfully employed in the degradation of different types of target proteins related to various diseases, including cancer, viral infection, immune disorders, and neurodegenerative diseases. PROTAC has various advantages in cancer therapy such as overcoming drug resistance and degrade traditionally “undruggable” protein target. At present, only 20-25% of the known protein targets can be targeted by using conventional drug discovery technologies. The proteins that lack catalytic activity and/or have catalytic independent functions are still regarded as “undruggable” targets. Moreover, large amount of oncoproteins, such as transcriptional factors, chromatin modulators and small GTPases, are hard to be directly targeted pharmaceutically. PROTAC is designed to target protein of interest (usually oncoprotein) for degradation by hijacking the endogenous E3 ligase and ubiquitin proteasome system. An example of an oral PROTAC drug is ARV-110, used for targeting Androgen Receptor for degradation, and has recently been approved by FDA for phase I clinical trial in treating patients with metastatic castration resistant prostate cancer in 2019.

In some embodiments, the molecules 110 comprise Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs). Mechanistically, a pair of ZFN monomers must bind the DNA, typically in a head-to-head configuration, by associating with DNA strands of opposite polarity. This catalyzes the DNA DSB (double strand breaks). TALENs are comprised of a DNA-binding domain composed by modular TALE repeats, fused with a FokI nuclease domain. Each TALE repeat is composed by 33-35 amino acids and recognizes a single nucleotide; specificity is determined by two hypervariable residues, known as Repeated Variable Diresidues (RVDs). Indeed, TALE repeats can be assembled together in a rather straightforward way to pair the desired DNA sequence, nucleotide by nucleotide. As for ZFNs, a pair of TALEN monomers is necessary to introduce a DSB. ZFN and TALEN have a limited range of targetable DNA sequences as ZFNs prefer G-rich sequences, while TALENs typically bind low G content sites strictly beginning with a T base.

It may be appreciated that the CRISPR/Cas9 system is more flexible, and targeting is usually easier and faster, as it suffices to design and synthetize a sgRNA complementary to the sequence(s) of interest. Multiple sequences may be targeted simultaneously, and no protein optimization is required. Because of its features, CRISPR/Cas technology is preferred in some instances over ZFNs and TALENs.

Biodistribution

A key feature of molecule transfer in the body involves biodistribution of the molecules. In order to successfully transfer the molecules to the cells, the molecules are to be in a desired location within the body at a desired concentration at a desired time in relation to the delivery of the energy for transfer.

In some embodiments, this is achieved with specialized infusion techniques. It may be appreciated that in some instances, infusion techniques are designed to provide a much higher concentration of molecules 110 in the vicinity of the target cells than would typically be used in viral based gene vectors or in vitro laboratory techniques. This may be achieved with a variety of methods. In some instances, the target cells are infused with a solution containing a high concentration of molecules. For example, in some embodiments, a solution containing 1-1000 times, such as 100-500 times, as many molecules as is used in viral based gene vectors or in vitro laboratory techniques. In some embodiments, 1-5 mg/ml of molecules is utilized. This is in contrast to more typical concentrations of 0.5-2 mg/ml. In other embodiments, a much larger volume of solution is provided in the vicinity of the target cells. This may increase the amount of molecules in the target location without increasing the concentration of the delivered solution. For example, a volume of solution 5 to 50 times, such as 10-25 times, the amount used in viral based gene vectors or in vitro laboratory techniques may be used. In some embodiments, 0.5-5 ml of molecules is utilized. This is in contrast to more typical volumes of 1-2 ml. It may be appreciated that in some embodiments, both an increased concentration and volume may be used. In some embodiments, increased volume of solution in the vicinity of the target cells is achieved by delivering solution at multiple locations around or near the target cells. In some instances, this is achieved with the use of an energy delivery device 102 comprising multiple tines, such as illustrated in FIG. 6 . In such embodiments, solution may be delivered to various locations through individual tines, such as surrounding a target tissue area. For example, these locations may be 0.5 mm-5 cm, particularly 1-25 mm, more particularly 5-20 mm, from the target tissue area. It may be appreciated that in some embodiments, different types of solution are delivered through different tines, such as different concentrations of solution, different volumes of solution and/or solution containing different types of molecules.

In some embodiments, the molecules 110 or other components of the solution are optimized to increase availability for transfer to the target cells in the body. For example, in some embodiments the molecules are altered to improve their dissolution and therefore distribution. In some embodiments, the molecules comprise linearized DNA, c3DNA or supercoiled DNA which may assist in cell transfer. In other embodiments, the DNA is conjugated with a targeting type addition, such as one that is drawn towards a specific target cell population (e.g. toward an antigen/protein on a cell). Typically, naked plasmid DNA has minimal cellular uptake because of its high negative net charge, very large molecular size, and susceptibility to enzymatic degradation. However, conjugation and similar methods can alleviate this. In some instances, lipid and polymer nanoparticles utilize cationic groups to bury and condense DNA plasmids for protection from nuclease degradation and neutralization of negative charges. Likewise, gold nanoparticles can be used to protect DNA plasmids from nuclease activity through conjugated PAMAM (polyamidoamine) polymers to the gold surface that create charge interactions with the negative phosphate backbone. In some embodiments, modified nucleotides are incorporated into specific sites of the plasmid, creating specific conjugation points that can be used for nanoparticle attachment. The nanoparticles are then a platform for a multitude of interactions of various polymers, specifically targeting aptamers for cellular uptake. For instance, a plasmid/nanoparticle complex conjugated to a CD44-aptamer promotes the targeted delivery of plasmid to breast cancer cells which highly express CD44 on the cellular membrane. Various aptamers may be used to direct the plasmid to specific cell types.

In some embodiments, genetic material or other components are combined with an addition that increases its ability to stay within a target tissue area. For example, in some embodiments, genetic material, such as DNA, is combined with a polymer such as polyethylene glycol which assists in the passage of the genetic material into the cell. In other embodiments, the genetic material or other components are combined in a solution that improves its dissolution or improves its ability to spread in a volume of tissue, such as a solution that changes the surface tension of fluid, such as a surfactant, glycol, dimethylsulfoxide, lipfectamine, cationic analgesics, etc.

In some embodiments, molecule 110 biodistribution is improved with the use of induced extravasation of fluid within a localized area in the body. Devices, systems, and methods to produce such extravasation may involve the delivery of energy, such as particular types of pulsed electric field energy or other suitable energy types. Such extravasation is typically from nearby vasculature, lymphatics, or other tissue which receives the energy. In some instances, the extravasation is edema or edema-like wherein capillaries leak fluid into the surrounding tissue. Edema occurs when an atypical volume of fluid accumulates in the tissues, either within cells (cellular edema) or within the collagen-mucopolysaccharide matrix distributed in the interstitial spaces (interstitial edema). The extravasation methods described herein focus on swelling of the extracellular matrix or interstitial edema. Naturally occurring interstitial edema may occur as a result of aberrant changes in the pressures (hydrostatic and oncotic) acting across the microvascular walls, alterations in the molecular structures that comprise the barrier to fluid and solute flux in the endothelial wall that are manifest as changes in hydraulic conductivity and the osmotic reflection coefficient for plasma proteins, or alterations in the lymphatic outflow system. However, the methods described herein induce the edema or extravasation by the delivery of specialized energy.

In some instances, the extravasation of fluid from the blood vessels carries molecules to the target tissue area that are delivered intravenously. In other instances, fluid leaks from the blood vessels and molecules are delivered regionally or locally, such as by injection, to the target tissue area. And in still other instances, extravasation is utilized alone without the delivery of molecules. Example improvements to the treatment therapy include, but are not limited to, conditioning the target tissue, increasing the availability of molecules, increasing the uniformity of the availability of molecules, increasing access to naturally restricted target tissues, creating larger treatment areas, and reducing potential undesired side effects, to name a few.

FIGS. 11A-11C illustrate various stages of an embodiment of an extravasation procedure. In this embodiment, the energy delivery device 102 comprises an elongate shaft 106 and an energy delivery body 108 disposed near the distal end of the elongate shaft 106. In this embodiment, the energy delivery body 108 is comprised of a single electrode and the distal tip 101 is configured to penetrate a portion of tissue in or near the target tissue area T. It may be appreciated that in other embodiments the energy delivery body 108 has an atraumatic tip and is delivered via a separate instrument that is able to penetrate tissue. As illustrated in FIG. 11A, the energy delivery body 108 is positioned within the target tissue area T near blood vessels BV, such as capillaries. In this embodiment, molecules 110 are delivered to the target tissue area T through the blood vessels BV, such as by IV administration. Such molecules 110 are particular to the treatment provided, such as genetic material for gene transfer.

FIG. 11A shows that only a few molecules 110 have entered the target tissue area but a significant quantity remains within the blood vessels BV. At least one dose of conditioning energy is then delivered to the target tissue area from the energy delivery body 108 as indicated by wavy lines 113, as illustrated in FIG. 11B. Typically, the conditioning energy comprises a specialized form of PEF energy, however it may be appreciated that other types of specialized energy may be used to cause the desired extravasation. In this embodiment, specialized PEF energy reversibly disrupts the fluid-barrier functional integrity of the endothelial cells within the blood vessels BV, such as by affecting the hydraulic conductivity and osmotic reflection coefficient for plasma proteins. This disruption causes the barrier to be less able to restrict the movement of fluid and macromolecules from the blood to the interstitium of the surrounding tissue. This causes extravasation and a flooding of the target tissue area with fluid and solutes, including molecules 110 from the blood vessels BV, as illustrated in FIG. 11C. The PEF energy typically disrupts the capillaries while causing minimal destruction of cells in the targeted area.

This process of extravasation may occur over a period of time, such as 5 seconds, or 30 seconds to 15 minutes. Therefore, in some instances it is desirable to begin delivery of the molecules 110 to the vasculature prior to delivery of the conditioning PEF energy to ensure maximum concentration and availability of the molecules 110 in the bloodstream. The period of extravasation and edema generation may vary in length depending on a variety of factors including the targeted organ, parameters used, and specific objectives of the therapy. For instance, molecules 110 that are not provided at a high systemic concentration may involve maximal extravasation effects prior to the therapeutic procedure. Likewise, molecules 110 that are heavily bioavailable may involve lesser extravasation effects prior to the therapeutic procedure. It is typically desired that the blood vessels BV be at their leakiest during the period that the concentration of the molecules 110 passing through these blood vessels BV are at the highest, thus providing the greatest extravasation of molecules 110 into the targeted tissue area interstitial environment.

The induced extravasation provides a variety of advantages. Example advantages include but are not limited to creating a larger treatment area, conditioning the treatment area to be more receptive to therapeutic treatment, increasing the availability of molecules, increasing the uniformity of the availability of molecules, increasing delivery of molecules to locations that are naturally restricted, such as across the blood brain barrier, and reducing the likelihood of potential side effects of the treatment.

In this embodiment, the molecules 110 are intended to be taken up by the cells of the target treatment area. In some embodiments, the induced extravasation alone is sufficient to increase the uptake of the molecules 110 by the cells of the target tissue area. In other embodiments, uptake of the molecules 110 is further facilitated with the delivery of therapeutic energy as will be described in further detail herein. It may be appreciated that, in some embodiments, the therapeutic energy is comprised of PEF energy having a different waveform than the conditioning PEF energy. It may be appreciated that in some embodiments, the therapeutic PEF energy is delivered with the same energy delivery body 108 positioned within the target tissue area. In other embodiments, a different device is used to deliver the therapeutic energy.

In some embodiments, the conditioning PEF energy has a waveform comprising monophasic, long-duration (>500 μs) pulses. FIG. 12A illustrates an example waveform of such PEF energy provided by an energy delivery algorithm 152 of the generator 104 used for inducing extravasation. In this embodiment, the waveform is comprised of a series of pulses 400, each having a pulse width 402 and amplitude (determined by the set voltage 404), wherein each pulse 400 is separated by a delay 406. In this embodiment, the pulse width is considered long duration and is greater than 500 microseconds. In this embodiment, the delay 406 between pulses 400 is in a range of 10 μs to 10 s, such as 1 ms, 500 ms, 1 second, 2 seconds, 5 seconds. In FIG. 12A, two pulses 400 are illustrated, however conditioning may be achieved with one, two, three, four, five, six, seven, eight, nine, ten, or more than ten pulses. In some embodiments, this PEF energy is not designed to induce uptake of the molecules 110 by cells within the target tissue area and therefore may utilize a range of pulse parameters (e.g. voltage, frequency, inter-pulse delays, etc.). However, it may be appreciated that in some embodiments, the treatment energy itself may be similar to that of FIG. 12A. In some embodiments, the waveform is biphasic, such as illustrated in FIG. 12B. Here, each pulse 400 is biphasic and has a pulse width 402 separated by a delay 406. In this embodiment, the pulse width 402 is again considered long duration and is greater than 500 microseconds. In this embodiment, the delay 406 between pulses 400 is in a range of 1 μs to 1 second, such as 1 μs to 10 μs, 10 μs, 1 μs to 100 μs, 100 μs, 1 μs to 250 μs, 250 μs, 1 μs to 500 μs, 500 μs, 1 ms, 2 ms, 5 ms or 1-5 ms, to name a few. It may be appreciated that in some embodiments, the pulses 400 reverse polarity such that some pulses 400 have a positive amplitude and some pulses 400 have a negative amplitude; such reverses in polarity may be symmetric or asymmetric. It may also be appreciated that in some embodiments, the pulses 400 are grouped by polarity. It may be appreciated that any suitable number of pulses may be present in each group, and each group may have the same or differing numbers of pulses. For example, six positive pulses may be followed by two negative pulses or four positive pulses may be followed by one negative pulse. Thus, various combinations can be made. Such groupings may be symmetrical or non-symmetrical.

It may be appreciated that in some embodiments, conditioning energy is delivered to the target tissue area that increases the cellular resistance of the target tissue area to eventual cell death. It is known that cells experiencing sub-lethal stresses will generate reparative and preventative responses to stress, in essence developing resistance to subsequent stresses of a similar or different nature, strengthening their resilience. For example, in some embodiments, the conditioning energy causes heat shock proteins (HSPs) to be released. HSPs are a family of proteins that are produced by cells in response to exposure to stressful conditions, such as conditioning energy as described herein. Although HSPs were initially described in relation to heat shock, HSPs are now known to also be expressed during other stresses including exposure to cold, UV light and during wound healing or tissue remodeling. Many members of this group perform chaperone functions by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by the cell stress. This increase in expression is transcriptionally regulated. The dramatic upregulation of the heat shock proteins is a key part of the heat shock response and is induced primarily by heat shock factor (HSF).

In some embodiments, pre-warming of the tissue or cells (prior to the delivery of the molecules 110) may start expression of heat-shock proteins, which play a role in cell injury, repair, and survival. In such embodiments, a warm solution, such as warm saline, may be injected to the treatment site wherein the molecules 110 are delivered after a wait period along with the delivery of energy. The wait period may be minutes, hours, or days after the delivery of the warming solution. In some embodiments, the wait period is 5-30 min, 1-2 hours, or 1-2 days.

In other embodiments, the tissue or cells are warmed with the use of the energy delivery body 108. In such embodiments, the energy is delivered at a controlled rate to maintain local temperature within a specific range, such as between 40-50C for a treatment of less than 10 minutes. It may be appreciated that in some embodiments, heat-shock proteins are triggered around approximately 41 C. Thus, sub-lethal pulsed electric field delivery may be used to encourage upregulation of heat-shock proteins and other damage-repair preparation prior to the stronger treatment pulsed electric fields. This encourages cell resilience to injury from the pulsed electric fields and improves the ability to transfer molecules to a meaningful number of cells without undesired excessive cell death.

Thus, in one embodiment, conditioning energy is delivered to the target treatment area which elevates temperatures in at least a portion of the treatment area, such as to 45 degrees Celsius. This induces extravasation of fluid to the area. A drug, gene, or other type of molecule is delivered via injection and benefits from the advantages of extravasation as describe herein. Therapy, such as therapeutic PEF energy is then delivered to the target treatment area. Since the cells of the treatment area were previously conditioned to resist cell death, a greater number of cells survive the treatment protocol. This is beneficial for gene therapy or other types of therapy which rely on cell survival.

Further examples of devices, methods and energy waveforms that provide such preconditioning, such as leading to extravasation, are provided in U.S. Provisional Patent No. 63/209,335, filed Jun. 10, 2021, entitled “INDUCED EXTRAVASATION BY ENERGY DELIVERY TO TISSUE” incorporated herein by reference for all purposes.

In some embodiments, biodistribution of the molecules is optimized by a controlled combination approach to delivery of the molecules 110 to the body. In some embodiments, delivery of molecules 110 by IV in combination with local injection provides a synergistic effect for enhanced transfer that is greater than IV alone or local injection alone.

Example 1: Delivery of enhanced green fluorescent protein (EGFP)-encoding plasmid DNA into the gastrocnemius hind muscle of balb/c mice with PEF energy. The plasmid DNA was administered to the mice in three ways 1) systemic injection (in the tail vein) of 20 μg plasmid in a volume of 200 μl saline equal to 1 mg/kg; 2) local injection of 200 μl of saline containing 200 μg of plasmid DNA inoculated in the gastrocnemius muscle using a drug delivery electrified needle; 3) combination of the previous routes of administration with first a systemic injection followed by a local administration prior/concomitant delivery of energy. Two different PEF energy delivery algorithms were used: 1) PEF algorithm A (Chopped Cycle Biphasic as will be described in later sections); and 2) PEF algorithm B (alternating DC as will be described in later sections). Depending on the energy delivery algorithm used, the local delivery of plasmid DNA was given 1 minute before the PEF algorithm B (8 seconds long protocol) or simultaneously with the PEF algorithm A (5 minutes long protocol) to ensure a slow and constant delivery of material while the energy is deposited in the tissue. To achieve constant and precise delivery of genetic material over the course of 5 minutes, an infusion pump was used to deliver 200 μl at the rate of 0.04 ml/min. During the delivery of genetic material and energy the mice were kept under gas anesthesia using isoflurane. Upon treatment, the mice were recovered and survived for 3 days before being euthanized and the gastrocnemius muscle collected. The muscle tissue was weighted, mechanically chopped with blades, lysed with RIPA buffer and sonicated to complete the homogenization process. The proteins were extracted from the homogenized tissue after ultracentrifugation and used to measure the EGFP protein by ELISA. The results were normalized to the weight of each tissue to obtain pg/mg concentration of EGFP protein in the samples. The results are illustrated in FIG. 13 . It may be appreciated that IV+IM=intravenous followed by local delivery. There were 5 samples measured for PEF algorithm A and 9 samples measured for PEF algorithm B. It may be appreciated that IM=local intramuscular delivery. There were 5 samples measured for PEF algorithm A and 10 samples measured for PEF algorithm B. It may be appreciated that IV=intravenous systemic delivery. There were 4 samples measured for PEF algorithm A and 4 samples measured for PEF algorithm B. FIG. 14 illustrates the results of FIG. 13 as average values. Thus, it can be seen that IV alone did not produce measurable transfer in this example while the protocol of IV+IM produced transfer that was greater than that would be expected by the combination of IV alone and IM alone. Further, it can be seen that, in this example, PEF algorithm B with the protocol of IV+IM produced the greatest level of transfer.

It may be appreciated that gene transfer is typically maximized if plasmid DNA is added before the application of the electric pulses which enable entry to the cell membrane and cause electrophoresis of DNA brought in contact with the cell membrane. Another postulated mechanism of gene electro transfer is that during electric field delivery the negatively charged DNA molecules move due to electrophoretic force and make contact with cell membrane in a larger number compared to free diffusion. Large plasmid DNA molecules form aggregates on cell membrane during electric pulses and enter the cell via endocytosis. For this additional reason it is desired to have high plasmid concentration at the site of transfer which is more challenging if the plasmid is administered systemically.

Energy

As mentioned previously, the energy delivery devices 102 deliver energy provided by the waveform generator 104 according to at least one distinct energy delivery algorithm 152. A variety of energy delivery algorithms 152 are provided that are specially designed for transfer procedures undertaken in the body. In some embodiments, the algorithm 152 prescribes a signal having a waveform comprising a series of pulses having delays therebetween. In such embodiments, the algorithm 152 specifies parameters of the signal such as energy amplitude (e.g., voltage), pulse width, delay period, and number of pulses, to name a few. It may be appreciated that the energy delivery algorithms 152 generate waveforms that maximize transfer yet avoid negative effects, particularly those specific to transfer in the body.

Conventional ex vivo techniques for electrogenetransfer typically involve energy from waveforms having pulses that involve a very long DC portion (e.g. 1-100 ms, such as 2-50 ms). Having long DC pulses of one polarity, or a sequence of pulses all delivered with the same polarity, increases the risk of ablation and size of a resulting ablation lesion. This is typically due to electrolysis, pH imbalance and net charge deposited into tissue. Electric currents flowing through biological matter produce a variety of effects in addition to transfer and potential Joule heating, such as electrolysis, ionthoporesis, and electro-osmotic flows. These can contribute to the formation of ablation.

In addition, when these imbalanced waveforms are used in the body, a variety of effects within the body can result. These imbalanced waveforms activate the motor neurons and skeletal muscle which can cause patient injury, patient pain and can negatively affect therapy outcome. Consequently, the energy delivery algorithms 152 described herein are configured to avoid these imbalances, thereby avoiding these deleterious effects.

FIG. 15A illustrates an example waveform provided by an energy delivery algorithm 152. In this embodiment, the waveform is comprised of a series of pulses 400, each having a pulse width 402 and amplitude (determined by the set voltage 404), wherein each pulse 400 is separated by a delay 406. In this embodiment, three pulses 400 are illustrated, however transfer may be achieved with one, two, three, four, five, six, seven, eight, nine, ten, or more than ten pulses. In some embodiments, the energy is delivered in such a monopolar fashion and the amplitude of each pulse or the set voltage 404 is 1-500V, 1-250V, 1-100V, 10-100V, 10-70V, 10-40V, 10-30V, 10-20V, 10V, 20V, 30V, 40V, 50V, 60V, 70V, 80V 90V, 100V, to name a few. The voltages used and considered may be the tops of square-waveforms, may be the peaks in sinusoidal or sawtooth waveforms, or may be the RMS voltage of sinusoidal or sawtooth waveforms.

In some embodiments, the pulse width is 1 ms, 2 ms, 5 ms, 10 ms, 20 ms, 30 ms, 40 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 1-20 ms, 1 ms-50 ms, 1 ms-100 ms, 2 ms-100 ms, 1-2 ms, to name a few. In some embodiments, the delay between pulses is 0.01-5 seconds, 0.01-0.1 seconds, 0.01-0.5 seconds, 0.01-1 second, 0.5 seconds, 0.5-1 second, 1 second, 1-1.5 seconds, 1-2 seconds, 0.5 to 2 seconds, 2 seconds, 1-3 seconds, to name a few. In some embodiments, the number of pulses is 1 pulse, 2 pulses, 3 pulses, 4 pulses, 5 pulses, 6 pulses, 7 pulses, 8 pulses, 9 pulses, 10 pulses, more than 10 pulses to name a few.

It may be appreciated that in preferred embodiments, the pulses 400 reverse polarity such that some pulses 400 have a positive amplitude and some pulses 400 have a negative amplitude. Such reversal diminishes charge imbalances which could lead to increased ablation and/or muscle contraction, as described above. It may be appreciated that in some embodiments the polarity alternates in an up, down, up, down fashion. For example, FIG. 15B illustrates an alternating DC waveform provided by an algorithm 152. Here, a positive pulse 400′ is followed by a negative pulse 400″ which then repeats in an alternating fashion. Again, each pulse 400′, 400″ has a pulse width 402 and amplitude (determined by the set voltage 404), wherein each pulse 400′, 400″ is separated by a delay 406. It may be appreciated that each pulse may differ in these parameters, such as having varying pulse widths 402, amplitudes 404 and delays 405. However, it is desired that the positive and negative on-times are balanced over time. In some embodiments, amplitudes 404 are in a range of 10-500V, such as 50-100V. In some embodiments, pulse durations 402 are in a range of 0.5-200 ms, such as 1-100 ms, 2-50 ms, or 20-30 ms to name a few. In some embodiments, the interpulse delays 406 are in a range of 10 ms-such as 200 ms-3 s or 500 ms-2 s. Optionally, the interpulse delays 406 may be synced with the ECG of the patient. In some embodiments, a treatment includes 1-100 pulses, such as 1-20 pulses or 1-10 pulses. These can culminate in total treatment durations of 0.5 to 500 ms (typically 20-200 ms). Table 1 below provides a variety of example parameter combinations to achieve transfer of molecules 110:

TABLE 1 Voltage (404), Pulse Duration Interpulse delay Pulse V (402), ms (406), sec count, # 1000 10 ECG 50 500 20 1 8 10 2 2 10 100 20 1 8 200 20 1 8 500 20 ECG 1 10 100 ECG 20 100 0.5 ECG 20

It may be appreciated that the algorithm B of FIGS. 13-14 produced a waveform having the shape of FIG. 15B. In that experiment, algorithm B produced the higher level of transfer under the conditions of both intramuscular injection alone and in combination with intravenous administration. Thus, this waveform has been demonstrated to retain significant transfer of affected cells while reducing or eliminating any potential lethal effects of the PEFs.

It may also be appreciated that in some embodiments, the pulses 400 are grouped by polarity. For example, FIG. 15C illustrates an example waveform provided by an energy delivery algorithm 152 wherein two pulses 400′ have a positive polarity followed by two pulses 400″ that have a negative polarity. It may be appreciated that any suitable number of pulses may be present in each group, and each group may have the same or differing numbers of pulses. For example, six positive pulses may be followed by two negative pulses or four positive pulses may be followed by one negative pulse. Thus, various combinations can be made. Such groupings may be symmetrical or non-symmetrical.

In some embodiments, one or more pulses 400 are “chopped”, partitioned or sectioned into an array of sectional-pulses of the same phase. Consequently, each pulse 400 is longer when “chopped” due to the inclusion of delays between the sectional-pulses that may be considered sectional-delays. FIG. 16 illustrates an embodiment of a waveform that is based on a conventional monophasic waveform (therefore considered the “base” waveform having base pulses), however it has been substantially altered to optimize transfer in the in vivo environment. Monophasic waveforms used in traditional gene therapy are DC pulses and typically have a pulse width of 1-50 ms, typically 20 ms. Here, a pulse similar to a monophasic waveform is shown (i.e. positive pulse 400′), however, here the positive pulse 400′ is chopped or partitioned into a plurality of sectional pulses 401 having sectional-delays 405 therebetween. Therefore, in this embodiment, the positive pulse 400′ has a pulse width in the range of 10-100,000 microseconds which is much longer than a monophasic DC pulse used in traditional gene therapy. However, the “on-time” for the pulse 400′ is the same as in the base waveform since the sectioning introduces delays rather than additional pulses. FIG. 16 also shows a negative pulse 400″ separated from the positive pulse 400′ by a delay 406. In this embodiment, the negative pulse 400″ is similar to the positive pulse 400′ however it has a negative polarity. Thus, the waveform of FIG. 16 overall is substantially different than a conventional monophasic waveform. The sectional-pulses break up the long driving forces during transfer of molecules into the cells so as to avoid muscle activation. It may be appreciated that electrophoresis is an important component of driving molecules toward and into cells during transfer of molecules 110 thereto. Typically, the timescale to drive electrophoretic motion in molecules of the size and molecular weight of molecules 110 described herein is more than the timescale for these transmembrane potentials to induce muscle activation. For example, neuron activation may occur with PEF energy delivery of 10 ms and skeletal muscle activation may occur with PEF energy delivery of 40 ms. Such activation is avoided by “chopping” the long driving force (pulse 402) into an array of sectional pulses 401 having short/brief pulse widths 403. This incrementally drives the molecules 110 into the cells wherein the interruptions (sectional-delays 405) permit relaxation of transmembrane potential charges, thereby preventing excitation of muscle. Thus, the small interruptions do not slow the driving force of the molecules 110 into the cells but they do slow the excitation of muscle.

It may be appreciated that in some embodiments, the pulses 400′, 400″ are relatively long, such as 10-100,000 μs or 100-50,000 μs and have delays 406 in a range of 100 μs-10 s, such as 1 ms-5 s or 50 ms-2 s, to name a few. These pulses 400′, 400″ may be chopped into a plurality of sectional-pulses 401 (such as up to 10,000 sectional-pulses 401, 10-2,000 sectional-pulses 401 or 20-1000 sectional-pulses 401, to name a few) wherein the sectional-pulse 403 durations are such as 0.5-20 μs, or 1-5 μs to name a few, and the sectional-delays 405 are 1-100,000 μs, such as 1-10,000 μs or 10-2,000 μs. In some embodiments, on-time does not exceed which may be considered a point of inflection for the initiation of muscle stimulation. In such embodiments, a delay 406 of 10 ms is desired between pulses 400′, 400″. Thus, the pulses 400′ 400″ and sectional pulses 401 of FIG. 16 are simply for illustration purposes to highlight the components of the waveform and are not drawn to scale for many embodiments as individual illustration of thousands of sectional-pulses would be prohibitively difficult. It may be appreciated that the pair 409 of pulses 400′, 400″ illustrated in FIG. 16 may be considered a cycle and is typically repeated, such as 1-1000 times, particularly 1-200 times, more particularly 1-100 times, more particularly 40-50 times, to name a few. A variety of repetitions may be utilized so long as the number of repetitions is not so small that muscle contractions coalesce (E>250 ms) and not so large that effects do not accumulate (E<10 s).

It may be appreciated that in some embodiments, voltage amplitudes are in a range of such as 50-250V or 50-100V, to name a few.

Table 2 below provides a variety of example parameter combinations to achieve transfer of molecules:

TABLE 2 # of Sectional- Sectional Voltage Sectional- Pulse Delay Delay (404), Pulses duration (405), (406), # of V (401) (403), μs μs μs Cycles 10 100,000 0.5 1000 1000 8 10 100,000 0.5 10 1000 1 1000 10,000 0.5 100 1000 1 50 400 5 10 5000 8 50 400 5 100 5000 8 50 400 5 1000 5000 8 10 10,000 10 1000 2000 1

FIG. 17 illustrates an embodiment of a waveform that is based on a biphasic AC waveform. Here, two pulses are shown, a positive pulse 400′ and a negative pulse 400″ separated by a delay 406 wherein the two pulses form a cycle. However, here the positive pulse 400′ is chopped into a plurality of sectional-pulses 401 having sectional-delays 405 therebetween. Likewise, the negative pulse 400″ is also chopped into a plurality of sectional-pulses 401 having sectional-delays 405 therebetween. Similar to the embodiments of FIG. 16 , these sectional-pulses break up the long driving forces during transfer of molecules into the cells so as to avoid muscle activation. Again, the small interruptions do not slow the driving force of the molecules 110 into the cells but they do slow the excitation of muscle. Generally, these embodiments change polarity more often than the embodiments of FIG. 16 . Likewise, in these embodiments the cycles 409 are typically grouped into packets (having inter-packet delays therebetween) and one or more packets are delivered during a treatment.

In some embodiments, the pulses widths 402 are somewhat shorter than those of FIG. 16 , such as 0.1-10,000 μs, 10-1,000 μs or 5-500 μs, but have similar delays 406 in a range of 100 μs-10 s, such as 1 ms-5 s or 50 ms-2 s, to name a few. These pulses 402 may be chopped into a plurality of sectional-pulses 401 (such as up to 1,000 sectional-pulses 401, 5-100 sectional-pulses 401 or 10-50 sectional-pulses 401, to name a few) wherein the sectional-pulse durations 403 are 0.05-50 μs, such as 0.5-20 μs, or 1-5 μs to name a few, and the sectional-delays 405 are 1-100,000 μs, such as 1-10,000 μs or 10-2,000 μs. Likewise, the pulses of FIG. 17 are simply for illustration purposes to highlight the components of the waveform and are not drawn to scale for many embodiments as individual illustration of thousands of sectional-pulses would be prohibitively difficult. It may be appreciated that the cycle 409 illustrated in FIG. 17 is typically repeated, such as 1-1000 times, particularly 1-100 times, more particularly 1-10 times, to name a few. A variety of repetitions may be utilized so long as the number of repetitions is not so small that muscle contractions coalesce (E>250 ms) and not so large that effects do not accumulate (E<10 s). In some embodiments, the cycles are grouped into packets having inter-packet delays therebetween. In some embodiments, a packet includes 1-100 cycles, such as 1-50 cycles, 1-20 cycles, 1-10 cycles, 1-5 cycles, etc. In some embodiments, a plurality of packets are delivered during a single treatment or transfer of molecules 110 to the target cells. In some embodiments, 1-100 packets are delivered, such as 1-50 packets, 1-20 packets, 1-10 packets, 1-5 packets, etc.

It may be appreciated that in some embodiments, voltage amplitudes are in a range of such as 10-1000V, 10-50V, 50-100V, 50-200V, 10y, 50V, 100V, 200V, 500V, 1000V, to name a few.

Table 3 below provides a variety of example parameter combinations to achieve transfer of molecules:

TABLE 3 Sectional- Sectional- Sectional- Number Number of pulse Pulse Delay Cycles of Voltage pulses duration delay (406), per packets (404), V (401), # (403), μs (405), μs μs Packet (411), # 1000 20 0.5 10 1000 1 1 100 20 0.5 10 1000 5 10 100 20 0.5 10 1000 10 5 10 10 10 1000 5000 5 10 50 2 5 50 100 20 10 200 25 5 1000 500 1 100 500 10 10 1000 1000 20 2

It may be appreciated that the algorithm A of FIGS. 13-14 produced a waveform having the shape of FIG. 17 . In that experiment, algorithm A produced a significant level of transfer under the conditions of both intramuscular injection alone and in combination with intravenous administration. Thus, this waveform has been demonstrated to retain significant transfer of affected cells while reducing or eliminating any potential lethal effects of the PEFs.

As mentioned, the pulses 400 may have differing characteristics, such as differing amplitudes (determined by the set voltage 404) and pulse widths 402. For example, FIG. 18A illustrates an example waveform provided by an energy delivery algorithm 152 wherein a first pulse 400′ having a first voltage 404′ and a first pulse width 402′ is followed by a differing second pulse 400″ having a second voltage 404″ and a second pulse width 402″. Here, the first voltage 404′ is higher than the second voltage 404″ and the first pulse width 402′ is narrower than the second pulse width 402″. Thus, the first pulse 400′ may be considered high and short in relation to the second pulse 400″ which may be considered low and long. In this embodiment, these pulses 400′, 400″ are then repeated. In some embodiments, the short-high pulse has a voltage amplitude 404′ in a range of 100-1000V and a pulse width 402′ in a range of 50 ns-1 ms, such as 50 ns-100 μs or 50 ns-10 μs. And, the low-long pulse has a voltage amplitude 404″ in a range of 5V-100V and a pulse width 402″ in a range of 1 ms-50 ms.

In some embodiments, such waveforms are considered multi-function waveforms. For example, in some embodiments, the short high voltage pulse creates the condition to allow the transfer and the long low voltage pulse drives the molecule into the cell, such as by electrophoresis. In some instances, molecules 110 approaching the cells, either driven by electrophoresis or simply moving closer to the cells by rotation, vibration or jiggling, “stick” to the cells and are pulled into the cell when the cell undergoes repair of its membrane. Thus, some molecules 110 may enter the cell by endocytosis, either during repair or during other cell states. In some embodiments, the short high voltage pulse is monophasic or biphasic. And, in some embodiments, the long low voltage is DC or alternating DC.

FIG. 18B illustrates a similar embodiment, however here the second pulse 400″ is negative. Thus, the first voltage 404′ is larger than the second voltage 404″, and in the opposite polarity. This provides charge balancing. In some embodiments, it is desired to balance the amplitude and on-time of the positive pulses with the negative pulses. This may be visualized by equating the areas of the pulses. For example, if the area of the positive pulse 400′ is equal to the area of the negative pulse 400′, the pulses are considered to be balanced. This allows for a variety of different shaped pulses. Such balancing reduces the net charge in either the positive or negative direction, thereby reducing muscle stimulation.

FIG. 18C illustrates another similar embodiment, however, here the pulses 400′, 400″ have the same polarity within a grouping or set, but the next set of pulses has the opposite polarity. It may be appreciated that the sets may be separated by a delay. Such delays are typically in the range of 1-3 seconds or may be synced with the heartrate of the patient. It may also be appreciated that transfer may be achieved by delivering one or more sets. In some embodiments, delivery of additional sets are predetermined, triggered (such as by one or more sensors) or applied at the decision of the user.

In some embodiments, the first pulse 400′ causes rotation of at least some of the molecules 110, such as pure rotation without translational effects. In some instances, this is achieved with a first pulse 400′ having a pulse width 402′ of 50 ns-1 ms, such as 50 ns-100 μs or such as having a native frequency at or above 1 GHz. In such embodiments, the second pulse 400″ has a pulse width 402″ of 1 ms-50 ms, such as having a native frequency below 1 GHz, such as in a range of 1 kHz-999 MHz. The second pulse 400″ causes a mixture of translational and rotational effects or causes pure translational effects. This combination of pulses 400′, 400″ accelerates movement of the molecules 110 toward the cells for transfer, such as harnessing rotational effects at the start of treatment to boost later translational effects. It may be appreciated that a plurality of first pulses 400′ and/or second pulses 400″ may be used in combination to tailor the effects.

As mentioned previously, in some embodiments, a series of pulses 400 are delivered in a packet, wherein packets are separated by inter-packet delays. FIG. 19 illustrates a packet 411 comprising a series of pulses 400; in this embodiment, the packet 411 is comprised of four pulses. Each pulse has a voltage 404 and pulse width 402. In this embodiment, each pulse is separated by a delay 406, likewise each packet is separated by an inter-packet delay 415. It may be appreciated that a packet 411 may be comprised of a series of identical pulses or of differing pulses. Likewise, the delays 415 between pulses may be regular or irregular. FIG. 20 illustrates a packet 411 comprising a series of pulses 400 having alternating polarities which may be considered a series of biphasic pulses, each biphasic pulse having a switch time delay 407. Here, each packet 411 is comprised of four pulses, each alternating in polarity, or two biphasic pulses.

It may be appreciated that in each of the embodiments described herein, the pulses (whether monophasic pulses, biphasic pulses, alternating DC pulses, etc.) may be grouped in packets 411. In some embodiments, packet delays are up to 20 seconds, such as 0.1-20 s, 0.1-10 s, 0.1-5 s, 0.1-2 s, 0.1-1 s, 0.5-1 s, 0.5-5 s, 0.1 s, 0.5 s, 1 s, 5 s, 10 s, etc. In some embodiments, the inter-packet delays are synced with an ECG.

In some embodiments, such as illustrated in FIGS. 21-22 , a series of high voltage, high frequency pulses are followed by a series of low voltage, low frequency pulses wherein the combination assists in transferring molecules 110 to target cells. For example, as illustrated in FIG. 21 , a first set of pulses 420 is delivered wherein the first set of pulses 420 comprises a plurality of high voltage, high frequency pulses, optionally in packets. Such pulses may prepare the cells for later transfer.

Examples of such high voltage, high frequency pulses are provided in U.S. Pat. No. 10,702,337, entitled “Methods, apparatuses, and systems for the treatment of pulmonary disorders” and PCT/US2020/028844, entitled “DEVICES, SYSTEMS AND METHODS FOR THE TREATMENT OF ABNORMAL TISSUE”, incorporated herein by reference, to name a few. Such pulses 420 may be used for ablation in other clinical applications, however for transfer or molecules 110 such pulses are adapted to prepare the cells for transfer. These pulses 420 are typically biphasic (having a positive pulse and a negative pulse which together form a cycle) and the cycles are grouped into packets. The cycle count is half the number of pulses within each biphasic packet. In some embodiments, the cycle count is set between 1 and 100 per packet, including all values and subranges in between. In some embodiments, the cycle count is up to 5 cycles, up to 10 cycles, up to 25 cycles, up to 40 cycles, up to 60 cycles, up to 80 cycles, up to 100 cycles, up to 1,000 cycles or up to 2,000 cycles, including all values and subranges in between. The packet duration is determined by the cycle count, among other factors. Typically, the higher the cycle count, the longer the packet duration and the larger the quantity of energy delivered. In some embodiments, packet durations are in the range of approximately 50 to 1000 microseconds, such as 50 μs, 60 μs, 70 μs, 80 μs, 90 μs, 100 μs, 125 μs, 150 μs, 175 μs, 200 μs, 250 μs, 100 to 250 μs, 150 to 250 μs, 200 to 250 μs, 500 to 1000 μs to name a few. In other embodiments, the packet durations are in the range of approximately 100 to 1000 microseconds, such as 150 μs, 200 μs, 250 μs, 500 μs, or 1000 μs.

Typically, there is a fixed rest period between packets. However, there may be a deliberate, varying rest period algorithm or no rest period may also be applied between packets. Table 4 provides example parameter values for such pulses 420.

TABLE 4 Example parameter combinations include: Packet Minimum # of Voltage Frequency duration Packets 3500 V 500 kHz 250 μs 200 5000 V  5 kHz 200 μs 10-20 6000 V 300 kHz 500 μs 100 3000 V 500 kHz 250 μs 25-50 2500 V 300 kHz 150 μs 100 2500 V 500 kHz 100 μs 50 2500 V 600 kHz 100 μs 20

In some embodiments, the first set of pulses 420 is followed by a delay 422 (such as 100 microseconds to 2 seconds) which is then followed by a second set of pulses 424. In this embodiment, the second set of pulses 424 is comprised a plurality of low voltage, low frequency pulses. FIG. 21 illustrates a first pulse 426 of the second set of pulses 424 lasting up to 10 microseconds followed by a delay 406 (e.g. up to 1 ms) and then a second pulse 428. In this embodiment, the second pulse 428 has opposite polarity to the first pulse 426, therefore the delay 406 may be considered a switch time delay 407. It may be appreciated that in some embodiments, there is no delay 406/407 between the pulses 426, 428. In some instances, the first set of pulses 420 prepares the cells for the transfer of molecules, such as putting them into a state that is more receptive to the molecules or the transfer process, the first set of pulses 420 starts the transfer process. The second set of pulses 424 then assist in transferring the molecules to the cells, such as the driving or pushing the molecules into the cells. Optionally, these sets of pulses 420, 424 may be repeated in a pattern.

FIG. 22 illustrates another example of a waveform having varied segments. Here, the first set of pulses 420 is delivered wherein the first set of pulses 420 comprises a plurality of high voltage, high frequency pulses, optionally in packets, such as described above in relation to FIG. 21 . Again, examples of such high voltage, high frequency pulses are provided in U.S. Pat. No. 10,702,337, entitled “Methods, apparatuses, and systems for the treatment of pulmonary disorders” and PCT/US2020/028844, entitled “DEVICES, SYSTEMS AND METHODS FOR THE TREATMENT OF ABNORMAL TISSUE”, incorporated herein by reference, to name a few, and described further hereinabove. In some embodiments, the first set of pulses 420 is followed by a delay 422 (such as 100 microseconds to 2 seconds) which is then followed by a second set of pulses 424. In this embodiment, the second set of pulses 424 is comprised a plurality of low voltage, low frequency pulses. In this embodiment, the second set of pulses 424 is comprised of a series of biphasic pulses having no switch time delay, wherein the second set of pulses 424 lasts approximately 100 microseconds to 5 milliseconds. Again, in some instances, the first set of pulses 420 prepares the cells for the transfer of molecules or starts the transfer process. The second set of pulses 424 then assist in transferring the molecules to the cells, such as the driving or pushing the molecules into the cells. Optionally, these sets of pulses 420, 424 may be repeated in a pattern.

It may be appreciated that rather than square waves, these pulses 400 may be sinusoidal or have other shapes. For example, FIG. 23 illustrates another embodiment of a pulse waveform. Here, each pulse is comprised of a plurality of increasing and decreasing voltages to form pyramid shapes.

It may be appreciated that in any of the embodiments described herein, the set voltage 404 may vary depending on whether the energy is delivered in a monopolar or bipolar fashion. In bipolar delivery, a lower voltage may be used due to the smaller, more directed electric field. The bipolar voltage selected for use in therapy is dependent on the separation distance of the electrodes, whereas the monopolar electrode configurations that use one or more distant dispersive pad electrodes may be delivered with less consideration for exact placement of the catheter electrode and dispersive electrode placed on the body. In monopolar electrode embodiments, the dispersive electrode may be comprised of a pad or any other recipient electrode. Typically, it functions as a dispersive electrode due to its size (large enough to prevent invoking effects locally where it is placed) and/or due to its placement (far enough away to avoid local effects and to not risk electrical arcing). However, in some embodiments, the dispersive electrode is small and may have some effects at its placement site, however such effects may be benign collateral effects. For example, the delivered molecules may not be present near the dispersive electrode so as to avoid transfer or any transfer in the area is inconsequential. In monopolar electrode embodiments, larger voltages are typically used due to the dispersive behavior of the delivered energy through the body to reach the dispersive electrode, on the order of 10 cm to 100 cm effective separation distance. Conversely, in bipolar electrode configurations, the relatively close active regions of the electrodes, on the order of to 10 cm, including 1 mm to 1 cm, results in a greater influence on electrical energy concentration and effective dose delivered to the tissue from the separation distance.

Energy delivery may be actuated by a variety of mechanisms, such as with the use of a button on the energy delivery device 102 or a foot switch operatively connected to the generator 104. Such actuation typically provides a single energy dose. The energy dose is defined by the number of pulses delivered and the voltage of the pulses.

FIGS. 24A-24C illustrate additional embodiments of waveforms provided by the algorithms 152. Here, rapid oscillations of high frequency are added to the pulses to provide additional push or rotation to the molecules 110 while the pulse overall drives the molecules 110 toward the cells. This might be particularly beneficial when a large molecule 110 or aggregate of molecules 110 has contacted a cell membrane but is having difficulty entering the cell. Such spikes in energy may provide the needed push to enter the cell. Referring to FIG. 24A, in this embodiment, a first pulse 400′ is provided having a pulse width 402 of first pulse width, such as followed by a second pulse 400″ having a second pulse width 402, such as 20 ms. Here, the pulses 400′, 400″ are separated by a delay 406, such as a delay of 5000 μs. In this embodiment, each of the pulses 400′, 400″ are in the positive direction and have a set voltage or primary voltage V1. In this embodiment, the voltage oscillates around the primary voltage V1 to create the spikes 413. Such oscillations are typically up to 25%, such as 0.1-25% or 1-25%, of the primary voltage, more typically up to 10%, such as 0.1-10% or 1-10%, of the primary voltage, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% of the primary voltage, or 15%, 20%, or 25% of the primary voltage. For example, with a 10% oscillation, a pulse 400′ having primary voltage V1 of 50V would oscillate between 55V and 45V to create the spikes 413 of 55V. Likewise, a pulse 400′ having a primary voltage V1 of 1000V would oscillate between 1100V and 900V with a 10% oscillation. Each spike 413 provides an additional push or rotation to the molecules 110 during translation. Thus, the spikes 413 due to the oscillations cause the pulses 400′, 400″ to appear as crown-like. It may be appreciated that pulses may include spikes 413 throughout the length of the pulse (i.e. pulse width 402), as illustrated, or the spikes 413 may occur at any point along the length of the pulse. Likewise, any number of spikes 413 may be present, including 100-10,000 spikes, 100-5,000 spikes, 500-5,000 spikes, 500-1000 spikes, to name a few. In some embodiments, the oscillations are at a frequency above 1 GHz which causes rotation of at least some of the molecules 110, such as pure rotation without translational effects. This combination of spikes 413 within the pulses 400′, 400″ assists in transferring the molecules 110 to the target cells, such as increasing push to or into the cells.

FIG. 24B illustrates a similar embodiment to FIG. 24A. Here, the each of the pulses 400′, 400″ are in the negative direction. Likewise, FIG. 24C illustrates the pulses 400′, 400″ in opposite directions. It may be appreciated that the waveforms described and illustrated herein are merely examples to illustrate various waveform concepts and are not intended to be an inclusive list of all possible waveforms. Likewise, these waveform concepts have been presented in a series of examples together with particular waveform features which may vary. Therefore, it may be appreciated that aspects, features and concepts of the waveforms may be utilized in various combinations and are not limited to the combinations described herein. For example, the concept of a “chopped” waveform may be applied to any of the base waveforms wherein the pulses of the base waveform are chopped as described herein. Likewise, the concept of the crown-like oscillations may be applied to any of the base waveforms wherein the pulses of the base waveform are altered to include the oscillations. Further, waveforms may be combined in various combinations, such as a portion of one waveform embodiment may be followed by a portion of a different waveform embodiment. Likewise, waveforms which induce extravasation may be utilized in combination with any of the waveforms disclosed herein or any variations.

Enhancements

The ability to deliver the molecules 110 to the tissue or cells may be altered with the use of a variety of enhancements. For example, in some embodiments, adjuvant materials are added to the body, such as added to a solution carrying the molecules, wherein the adjuvant materials render the cells more susceptible to small molecule or macromolecule transport. Example adjuvant materials include polymeric nanoparticles, liposomes, PEGylated liposomes, lipofectamine, cell-penetrating peptides (CPC), dimethyl sulfoxide (DMSO), cholesterol, or other materials known to interact with cell membrane fluidity and mechanics. In some embodiments, the adjuvant material is injected, and the injection pressure is chosen or adjusted to enhance uptake of the molecules 110 by the cells.

In other embodiments, the tissue or cells are warmed or cooled to alter their ability to successfully receive the molecules 110. For example, in some embodiments, the cells are warmed or cooled, such as by warming or cooling a solution carrying the molecules 110, to invoke better transfer efficiency or improved likelihood of cell survival following energy delivery. In some embodiments, warming of the cells may increase membrane fluidity and therefore increase acceptance of the molecules 110. In other embodiments, cooling of the cells may increase rigidity and potential formation of “cracks” which increase acceptance of the molecules 110.

In other embodiments, pre-warming of the tissue or cells (prior to the delivery of the molecules 110) may start expression of heat-shock proteins, which play a role in cell injury, repair and survival. In such embodiments, a warm solution, such as warm saline, may be injected to the treatment site wherein the molecules 110 are delivered after a wait period along with the delivery of energy. The wait period may be minutes, hours or days after the delivery of the warming solution. In some embodiments, the wait period is 5-30 min, 1-2 hours or 1-2 days.

In some embodiments, the tissue or cells are warmed with the use of the energy delivery body 108. In such embodiments, the energy is delivered at a controlled rate to maintain local temperature within a specific range, such as between 40-50C for a treatment of less than 10 minutes. It may be appreciated that in some embodiments, heat-shock proteins are triggered around approximately 41 C. Thus, sub-lethal pulsed electric field delivery may be used to encourage upregulation of heat-shock proteins and other damage-repair preparation prior to the stronger treatment pulsed electric fields. This encourages cell resilience to injury from the pulsed electric fields and improves the ability to transfer molecules to a meaningful number of cells without undesired excessive cell death.

As mentioned previously, the processor 154, among other activities, modifies and/or switches between the energy-delivery algorithms, monitors the energy delivery and any sensor data, and reacts to monitored data via a feedback loop. In some embodiments, the processor 154 is configured to execute one or more algorithms for running a feedback control loop based on one or more measured system parameters, one or more measured tissue parameters, and/or a combination thereof. In some embodiments, the parameter includes temperature so that temperature is able to be maintained within a specific range by controlling the cadence of energy delivery. This may be useful for enhancing cell uptake, immune response, overall safety, etc.

It may be appreciated that enhancements may be applied before, during or after delivery of the molecules 110 and/or before, during or after delivery of the treatment energy. In some embodiments, adjuvant material is administered to the patient at a desired interval during a multi-function waveform, such as between a short high pulse and a long low pulse of an asymmetric waveform. This may assist in driving or pushing the adjuvant material into the cells.

It may be appreciated that in some embodiments, isotonic or hypertonic saline solution is delivered to the treatment site to adjust local tonicity.

Sensors

As mentioned previously, in some embodiments, the energy delivery device 102 includes one or more sensors that can be used to determine pressure, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties (coherence, echogenicity, fluorescence), electrical or light permittivity, and/or conductance, to name a few. In some embodiments, one or more of the electrodes act as the one or more sensors. In other embodiments, the one or more sensors are separate from the electrodes. Sensor data can be used to plan the therapy, monitor the therapy and/or provide direct feedback via the processor 154, which can then alter the energy-delivery algorithm 152.

A. Pressure Sensing

It may be appreciated that cells typically respond to mechanical stimuli. One such type of mechanical stimuli is pressure. In general, hydrostatic pressure is determined by the interstitial fluid volume and the general compliance of the targeted tissue interstitium. Important to note that this is variable for tissue types as some organs are encased within a more rigid/less compliant structure (e.g., brain, kidney, etc.), while others are more free to expand/contract (e.g., lungs, muscle, skin, etc.). While, increasing hydrostatic pressure can increase permeability of local cell membranes, excessive amounts tissue interstitial pressure can certainly lead to tissue damage and cell death It has been shown that mammalian cells undergo varying forms of apoptosis at pressures between 200-300 MPa and succumb to a more immediate cell death at pressures between 300-400 MPa.

Thus, it is often beneficial to be mindful of the pressure that the target cells are enduring, particularly during injection of molecules 110 when local injection pressure may affect the target cells. Consequently, in some embodiments, the energy delivery device 102 includes a pressure sensor, typically located so as to monitor the local injection pressure.

In some embodiments, feedback from the pressure sensor is used to prevent harmful pressures from being reached. For example, pressure measurements from the sensor may be displayed for the user, one or more algorithms 152 may be altered based on the measurements, and/or an alert or shutoff may occur when the pressure reaches a predetermined threshold.

However, hydrostatic pressure plays an important role in permeabilization of cell membranes. In some instances, a desirable extracellular pressure increase will facilitate the transfer of molecules 110, such as exogenous DNA, into the cells. In addition, hydrostatic pressure often plays an important role in distribution of the injected molecules 110 withing the tissue. Thus, in some embodiments, pressure measurements (e.g. external pressure measurements taken at the proximal end of the device 102) may be utilized by the user as a metric to ensure optimized transfer conditions. In such embodiments, pressure can be increased or decreased to fall within a desired predetermined pressure range. In some embodiments, pressure is measured before injecting the molecules 110 (minimal or null injection flow rate) to identify the basal intracellular pressure (Pi=P1|Q1=0). Considering a defined target differential pressure (Pd=Pi−Pe) optimal to achieve transfer and knowing the hydrostatic pressure of the system to the solution (Rapplicator), the inflow rate (Q1) is adjusted until the pressure increases the desired pressure difference (P1=Pi+Pd+Q1*Rapplicator). Thus, the injection rate and treatment application initiation can be optimized to achieve desired outcomes.

When delivery methods include induced extravasation, as described above, pressure sensor measurements may be utilized to monitor edema levels, such as before, during and/or after injection of molecules 110 and treatment energy. Thus, inducement of extravasation can be adjusted to reach desired edema levels at particular times throughout a treatment protocol.

A variety of pressure sensors 200 may be used. In some embodiments, the pressure sensor 200 is disposed along a distal tip of the energy delivery device 102, such as illustrated in FIG. 25A. Here, the energy delivery device 102 comprises an energy delivery body 108 having a needle shape, wherein the energy delivery body 108 is at least partially covered by an insulative sleeve 202. As illustrated, molecules 110 are passed through the energy delivery body 108 to the nearby target tissue area. This allows the pressure sensor 200 to monitor the pressure during the injection of the molecules 110 and application of treatment energy. In other embodiments, the pressure sensor 200 is disposed along the distal end of the energy delivery device 102 but proximal to the tip, such as illustrated in FIG. 25B. Here, the pressure sensor 200 is disposed along the insulative sleeve 202 that at least partially covers the energy delivery body 108. Having a relative pressure measurement at the tissue level allows the user to understand the distribution of the injected molecules 110 within the tissue. Considering the injected solution at a known pressure and flow rate (values able to be easily measured near the proximal end of the device 102), an additional relative measurement at the distal end will provide the information to understand the spatial distribution of the molecules along the target tissue as well as the temporal pressure profile.

In some embodiments, the pressure sensor 200 comprises a strain-gauge transducer. Strain-gauge transducers are typically characterized by exhibiting a change in their form of output in response to the measurand (i.e., strain, electrical resistance, or wavelength). Sensitivity is determined by the relative change of resistance with respect to length.

In other embodiments, the pressure sensor 200 comprises a diaphragm displacement sensor. Diaphragm displacement sensors are based on micro-electromechanical systems technology, in which the sensors have a bendable flat surface (diaphragm) over a sealed cavity. The diaphragm bends or deforms in response to the change in pressure. The resultant form of output can be capacitance based or piezoelectric transducer based. In some embodiments, a sensor 200 is placed at the distal tip of the energy delivery device 102, while its corresponding diaphragm is disposed proximal to the sensor 200, thus enabling a measurement of the pressure drop across the distance therebetween. In instances where such placement of a sensor is difficult, such as due to size restrictions, a pressure sensing optical fiber may be preferred.

In some embodiments, the energy delivery device 102 includes an expandable member 204 disposed along its distal end, such as illustrated in FIG. 25C. Here, the expandable member 204 is mounted on an insulative sleeve 202 that at least partially covers the energy delivery body 108 having a needle shape. Such positioning of the expandable member 204 can assist in preventing reflow of the solution of molecules 110 back up the pathway created by inserting the energy delivery device 102. This may improve the pressure distribution within the target tissue and therefore overall transfer the molecules 110. The expandable member 204 may also prevent movement of the needle-like energy delivery body 108 during delivery of the PEF energy. In some embodiments, a pressure sensor 200 mounted on the expandable member 204 monitors the pressure within the expandable member 204. This ensures proper inflation of the expandable member 204. Further, in some embodiments, the pressure sensor 200 monitors tissue pressure during infusion of molecules 110 and/or PEF energy delivery.

B. pH Sensing

It may be appreciated that cells also typically respond to biological stimuli. One biological stimulus that plays an essential role in cell viability is pH. Assuming stable pH, relatively minor changes within the pH can potentially affect virtually all cellular process, including metabolism, membrane potential, cell growth, movement of substances across the cell membrane, state of polymerization of the cytoskeleton and the ability to contract in muscle cells.

In some embodiments, a pH sensor is provided (e.g. disposed along the energy delivery device 102, such as near its distal tip) to monitor local pH. Measurements of pH can be used to alert the user or alter one or more algorithms 152 to avoid harmful acidity/alkalinity from being reached, but more importantly could also be optimized and utilized as a metric for optimal transfer conditions/setting.

C. Temperature Sensing

Another biological stimulus that plays an essential role in cell viability is the temperature. Relatively minor changes in temperature can potentially affect virtually all cellular process, including metabolism, membrane potential, cell growth, movement of substances across the cell membrane, state of polymerization of the cytoskeleton and the ability to contract in muscle cells. In some embodiments, a temperature sensor is disposed along the energy delivery device 102 to monitor the local temperature. This will enable the ability to monitor and maintain local temperature within a normal, physiologic range, while diminishing or ensuring no thermal damage during energy delivery. Further, in some embodiments, temperature sensing is used in combination with moderate cooling/heating associated with the delivery of the molecules 110 to the target site and ultimately improve transfer, as described previously. It may be appreciated that a variety of types of temperature sensors may be used such as thermocouples, resistance temperature detector sensors, fiberoptics, etc.

Transfer and Ablation

It may be appreciated that in some embodiments the devices, systems and methods described herein may be adapted to provide ablation in addition to transfer of molecules 110. This may be particularly useful when treating tumors in the body. In such instances, the energy delivery device 10 is configured to access the tumor, such as percutaneously or endoluminally. The generator 104 includes one or more algorithms 152 that deliver PEF energy having waveforms that provide ablation in particular locations within the target tissue and provide transfer of molecules to other locations within the target tissue. In some embodiments, the PEF energy creates various zones of treatment extending radially outwardly from the energy delivery body 108. For example, a zone closest to the energy delivery body 108 (i.e. a central zone) endures immediate cell death, such as via necrosis, and a zone surrounding the central zone (i.e. a peripheral zone) receives transfer of molecules 110, such as for gene therapy.

It may be appreciated that some of the PEF energy waveforms described herein above may be adapted to provide ablation in addition to transfer of molecules 110. For example, in some embodiments, the alternating DC waveform illustrated in FIG. 15B is configured to provide ablation in addition to transfer of molecules 110 by modifying various parameter values. Table 5 below provides a variety of example parameter combinations to achieve ablation in a central zone and transfer of molecules 110 in a peripheral zone:

TABLE 5 Voltage Pulse Duration Interpulse delay Pulse (404), V (402), ms (406), sec count, # 2000 10 ECG 100 1000 50 1 1 1000 20 1 8 500 100 1 10

In some embodiments, the “chopped” waveform of FIG. 16 that alternates in polarity is configured to provide ablation in addition to transfer of molecules 110 by modifying various parameter values. Table 6 below provides a variety of example parameter combinations used with this waveform to achieve ablation in a central zone and transfer of molecules 110 in a peripheral zone:

TABLE 6 Sectional- Number of Sectional Voltage Pulse Sectional- Pulse Delay Cycles (404), duration Pulses delay (406), per V (403), μs (401), # (405), μs ms Packet 6000 1 1,000 100 1000 10 1000 5 400 10 1000 5 1000 5 400 1000 1000 5 500 10 200 500 500 10 100 1 20,000 100 500 10 100 2 10,000 100 500 10 100 2 5,000 100 500 10 500 10 10 1000 1000 100 3000 10 200 1000 500 100 3000 10 200 1000 500 10 3000 5 400 1000 500 10 3000 1 2000 1000 500 10

In some embodiments, the “chopped” biphasic waveform of FIG. 17 that alternates in polarity is configured to provide ablation in addition to transfer of molecules 110 by modifying various parameter values. Table 7 below provides a variety of example parameter combinations used with this waveform to achieve ablation in a central zone and transfer of molecules 110 in a peripheral zone:

TABLE 7 Sectional- Number of Number pulse Sectional- Sectional- Delay Cycles of Voltage duration pulses delay (406), per packets (404), V (403), μs (401), # (405), μs μs Packet (411), # 6000 5 25 1000 1000 1 100 6000 2.5 10 1000 500 10 100 1000 5 100 1000 1000 5 50 1000 10 10 2000 100 200 100 1000 10 10 2000 100 50 100 500 1 500 10 2000 10 250 100 10 100 500 500 20 100 3000 10 10 1000 1000 10 10 3000 7.5 15 1000 500 5 100 3000 0.5 20 10 500 20 100 3000 0.5 10 1000 1000 20 200 4500 5 4 1000 1000 5 100

As mentioned previously, in some embodiments, the energy delivery device 102 comprising a shaft 106 having an energy delivery body 108 near its distal end, wherein the energy delivery body 108 comprises a plurality of tines 600. Likewise, referring to FIG. 6 , in some embodiments the first section 106 a acts as an energy delivery body 108 and one or more tines 600 act as energy delivery bodies 108. Each of the different energy delivery bodies 108 may deliver the same or different types of energy; likewise, the energy delivery bodies 108 may act in groups. In particular, in some embodiments the first section 106 a delivers energy that ablates tissue and one or more tines 600 deliver energy that transfers molecules to cells of the tissue. It may be appreciated that in other embodiments, particular tines deliver energy that ablates tissue while other tines deliver energy that transfer molecules to cells of the tissue. It may also be appreciated that in some embodiments the same tines are used to deliver different types of energy at different times. Likewise, it may be appreciated that in some embodiments the same tines are used to deliver molecules 110 or energy at different times. And in some embodiments, the same tines can be used to deliver molecules 110 and energy at the same time. These embodiments may be particularly useful when treating undesired tissue such as a tumor, wherein the tumor is ablated and molecules 110 are delivered therearound to ensure complete removal of the tumor.

Additional Clinical Applications

A. Delivery to Heart Tissue

FIG. 26 illustrates a method of delivering molecules 110 to cells within the heart H with the use of an energy delivery device 102. In this embodiment, the energy delivery device 102 comprises has an elongate shaft 106 and an energy delivery body 108 disposed near its distal tip. In addition, the shaft 106 includes one or more delivery ports 702 located a distance proximal to the energy delivery body 108 through which the molecules 110 are delivered. In some embodiments, the one or more delivery ports 702 are positioned so as to reside within the aorta A, above the aortic valve AV, when the energy delivery body 108 is positioned within the left ventricle LV. In the embodiment of FIG. 26 , the one or more delivery ports 702 are disposed above the aortic valve AV, in the vicinity of the coronary artery take-offs, while the energy delivery body 108 is positioned near a wall of the left ventricle LV. This allows the molecules 110 to be delivered to the coronary artery take-offs, where the molecules 110 are able to enter the coronary artery circulation. FIG. 27 illustrates the coronary arteries CA and the position of the coronary artery take-offs in relation to the aorta A and the aortic valve AV. Thus, delivery of the molecules 110 to the aorta A, above the aortic valve AV, allows the molecules 110 to enter the coronary artery circulation so as to reach various tissues within the heart H. The molecules 110 are largely prevented from entering the left ventricle LV due to the one-way aortic valve AV. The energy delivery body 108 positioned within the heart H, such as within the left ventricle LV, is able to deliver energy to wall of the heart H so that molecules 110 circulating nearby through the coronary arteries CA are able to enter the tissue cells of the wall of the heart H.

It may be appreciated that the energy delivery device 102 may have a variety of configurations so as to deliver energy to the heart H either at one location or at multiple locations, either simultaneously or in a pattern. Likewise, additional delivery devices may be used to deliver energy and/or molecules 110 to various locations within or near the heart H. This may be useful when delivering energy to portions of the heart H accessible most easily accessible from various chambers.

It may be appreciated that in some embodiments the molecules 110 are deliverable from the distal end of the energy delivery device 102, such as within the left ventricle LV so as to reside near the wall to which energy is delivered or through a penetration into the wall so that the molecules 110 are delivered into the wall prior to or during energy delivery. It may be appreciated that any combination of molecules 110 and energy delivery may be utilized. In any of these scenarios, the molecules 110 are delivered to the tissue so that the cells are able to uptake the molecules 110 upon energy delivery.

B. Delivery to Extramedullary Hematopoiesis Masses

Hematopoiesis is the production of all types of blood cells including formation, development, and differentiation of blood cells. Prenatally, hematopoiesis occurs in the yolk sack, then in the liver, and lastly in the bone marrow. However, in some patients, hematopoiesis occurs outside of the bone marrow, either naturally or pathologically (e.g. in chronic anemic conditions such as thalassemia, sickle cell disease, and myeloproliferative disorders, in patients with classic myeloproliferative disorders, particularly chronic idiopathic myelofibrosis, and in other myeloproliferative disorders with “secondary myelofibrosis,” such as polycythemia vera and, rarely, essential thrombocythemia). Less frequently, it can also be seen in patients with myelodysplastic/myeloproliferative diseases (e.g., chronic myelomonocytic leukemia) or in myelodysplastic syndromes. This is considered extramedullary hematopoiesis (EMH) and refers to hematopoiesis occurring outside of the bone marrow. Extramedullary hematopoiesis is often observed in the spleen and liver, but is also seen in other sites, such as lymph nodes, lung, serosal surfaces, thorax, adrenal glands, urogenital system, skin, and retroperitoneal and paraspinal spaces.

It is hypothesized that EMH is a compensatory response to an increased need for blood production. Ineffective erythropoiesis in patients with hemoglobinopathies drives extramedullary hematopoietic (benign) tumor formation or masses in several parts of the body. Such masses can reach a few centimeters in dimensions. Since such masses are generally benign, the masses are seldomly removed. In some embodiments, cells in such masses, such as CD34+ cells (hematopoietic stem cells, HSC), are targeted for transfer of molecules 110 utilizing the PEF energy, devices and methods described herein. Such locations, particularly within the liver or spleen are relatively easy to reach, and the immune cells produced after treatment, such as editing of a genetic mutation, would be released in the body to fix or mitigate the corresponding disease.

C. Delivery to Bone Marrow

In some embodiments, transfer of molecules 110 to stem cells takes place directly in the bone marrow of the patient, utilizing the PEF energy, devices and methods described herein. In some embodiments, the energy delivery device 102 is configured to access the bone marrow itself and in other embodiments the energy delivery device 102 is configured to be advanced into the bone marrow after an accessory device provides access. In some embodiments, hematopoietic stem cells are targeted for transfer. In such embodiments, the iliac crest (in the pelvis) is perforated to access target cells within the bone marrow. The iliac crest is a bone rich in hematopoietic stem cells, is easily accessible, and is routinely used to access bone marrow for bone marrow donation. Perforation creates a hole through which the energy delivery device 102 is advanceable so as to deliver the PEF energy. As mentioned, molecules 110 may be delivered through the energy delivery device 102 or through various other routes. Ultimately, the molecules 110 transfer to the target cells with the assistance of the PEF energy. Thus, direct (in vivo) delivery of molecules 110, such as gene editing material, in hematopoietic stem cells (HSC) of the bone marrow to cure genetic hematological disorders.

Direct delivery of molecules 110 to bone marrow is superior to ex vivo methods. In ex vivo methods, hematopoietic stem cells (CD34+ cells) are isolated from mobilized peripheral blood. Mobilized peripheral blood is the blood circulating throughout the body that has been treated with a mobilizing agent. The term “mobilization” refers to the process whereby stem cells are encouraged to migrate from the bone marrow to the peripheral circulation where they can be collected via leukapheresis. The mobilizing agents cause a large number of hematopoietic stem and progenitor cells to be mobilized from the bone marrow and extravasate into the bloodstream. Due to the nature of these agents and the complexity surrounding donor recruitment and collection, donors must be monitored closely, and collection must be managed continuously to ensure both donor safety and successful collection.

In addition, expansion of stem cells in vitro using growth factor can transform the stem cells into cancer cells. Indeed, some sickle cell anemia patients in CRISPR clinical trials have developed hematological cancers after the reinfusion of cells. Reinfusion of edited stem cells into the patient will require rounds of chemotherapy to suppress the immune system and allow them to graft otherwise the body will reject them.

In bone marrow, only 5-10 percent of CD34+ are HSC. Ex vivo CD34+ selection does not eliminate T cells entirely and this has implications for allogeneic transplantation, as graft-versus-host disease (GvHD)-free transplantation requires pure HSCs without T cell contamination. About 95% of endogenous HSCs are in cell cycle stage G0. After classic cyclophosphamide/granulocyte colony-stimulating factor (G-CSF) mobilization, all marrow HSCs enter cell cycle and expand, and upregulate the expression of the macrophage “don't eat me” signal, CD47. They enter the bloodstream at G0/G1, home to the bone marrow through interactions between HSC integrin α4β1 and sinusoidal endothelial vascular cell adhesion molecule, cross-fields of macrophages, and home to the CXCL12 niche through the HSC CXCR4 receptor. When HSCs are cultured in vitro with factors, they enter the cell cycle, entering G1 and at ˜30 h entering S phase. HSCs in S/G2/M lose the ability to home to marrow when injected intravenously. At 2-3 days of culture, the majority of the HSCs have become progenitor cells that cannot self-renew. Thus, only a small fraction of CD34+ cells to begin with are HSCs, and the HSCs within them that are in cycle home poorly.

In addition, the biology and environment of HSCs have implications for viral vector HSC gene modification. Unlike cells, viral vectors cannot home to the bone marrow from blood, and it is unlikely that they can traverse marrow vessels to enter HSC niches. Current methods of mobilization will provide both vectors and G0/G1 HSCs in the blood only for brief intervals, and even mobilized HSCs represent a rare population. Although viral envelope proteins can promote fusion with HSC, allowing viral vectors to release their cargo into the cell, vectors for gene therapy will also need HSC-specific binding sites and off-target delivery, and integration could still be a problem. Nevertheless, producing lentiviral vectors with envelope ectodomains that bind HSC-enriched markers could enable in vivo gene transfer, and experimental manipulation of the mobilization regimen could prolong the residence time vector accessibility of HSC in the blood. None of these strategies contemplate the molecule 110 delivery and subsequent effects, such as gene editing, directly in the bone marrow as described herein.

It may be appreciated that the CRISPR/Cas9 system is a preferred system for gene editing. The CRISPR/Cas9 system may be utilized for gene editing at various locations within the body, including the bone marrow, with the PEF energy, devices and methods described herein. This is in contrast to conventional tools for gene editing in HSCs, such as a lentiviral vector that expresses both Cas9 and the guide RNA which is not optimal because of low titers, persistent Cas9 expression after transduction, and difficulties in achieving transduction. Similarly, conventional electroporation with Cas9 plus guide RNA ribonucleic particles works efficiently in vitro, but can be associated with a high level of toxicity and is not available as an in vivo application.

D. Delivery to Mesenchymal Stem Cells

In some embodiments, transfer of molecules 110 to mesenchymal stem cells takes place directly in the body, such as in the bone marrow or adipose tissue of the patient, utilizing the PEF energy, devices and methods described herein. Mesenchymal stem cells (MSCs) are a desirable therapeutic target for many diseases, especially those related to the musculoskeletal system. MSCs are adult stem cells that can differentiate into cells of the mesoderm-lineage, including osteoblasts, chondrocytes and adipocytes. MSCs are rare, non-hematopoietic progenitor cells first isolated from the bone marrow (BM). Though mainly found in BM and adipose tissue, examples of isolation from peripheral blood, umbilical cord blood, synovial membranes, deciduous teeth, amniotic fluid and perivascular regions exist.

MSCs have been found to be both hypoimmunogenic and immunomodulatory, can home to damaged tissues and depend on secretion of bioactive molecules to initiate healing in repair processes. MSCs express an assortment of chemokine receptors that allow for their migration in response to the chemokine-attractive gradients generated by the inflamed injured site. Therefore, genetic modification of these cells, such as with the use of the devices and methods described herein, could be used to treat diverse diseases from cancer to cardiovascular or skeletal diseases.

In vivo transfer of molecules 110 to MSCs overcomes many of the drawbacks of ex vivo transfer. To begin, long-term cell culture is avoided which eliminates the consequent effect of increasing heterogeneity of the cell population. An increasingly diverse population of cells makes it hard to clearly define the functional population and the mechanisms responsible for any detectable effect. In addition, long-term culturing can also lead to down regulation of functional surface proteins, such as CXCR4, and long-term ex vivo expanded cells show reduced migration and homing potential in vivo. Thus, any problems related to transferring of results from the bench to the bedside is avoided. Finally, along with concerns of serum-dependent growth for transplant into humans, cultured MSCs can acquire mutations and become transformative as in the case with the formations of sarcomas from transplanted MSCs as seen in mice. This is also avoided with in vivo transfer.

It is desirable to transfer molecules 110 to MSCs in the microenvironment in which they reside. The microenvironment where MSCs reside, whether it is perivascular, in the BM or in another area, has a substantial effect on the homing, differentiative and regenerative capabilities of MSCs. The surrounding cells and extracellular milieu also seem to influence the surface proteins and epitopes which may be used as identifying markers.

It may be appreciated that MSCs may be targeted directly in vivo for gene editing or gene therapy. These manipulations of MSC benefit the following diseases.

Enhance engraftment of hematopoietic stem cells transplant. MSCs were first isolated from BM, a key site of hematopoiesis in which MSCs are now thought to play a role. Hematopoietic stem cells (HSCs) require stromal supporting cells for proper differentiation and for the maintenance of the quiescent state within the endosteal niche of the BM and both supporting functions can be carried out by the MSC-progeny, osteoblasts. This has been the basis for the use of MSCs in combination with HSC transplantation in the hopes of enhancing engraftment and proliferation of donor HSC. Genetically modified MSC at the site of transplantation would improve engraftment.

Hypophosphatasia: Hypophosphatasia is a disease characterized by reduced mineralization of the bone due to inactivating mutations in tissue non-specific alkaline phosphatase (ALP) from osteoblasts and chondrocytes. Transplanted MSC-differentiation into osteoblasts could be an effective treatment for delivery of a population of cells able to produce normal ALP enzyme. Whole BM transplants have been used to treat infants suffering from hypophosphatasia in combination with cultured osteoblast cells. However, local (in the BM) transfection of ALP enzyme to MSC using the devices and methods described herein would improve outcomes.

Osteogenesis imperfecta: Osteogenesis imperfecta (OI) is a group of at least nine genetic disorders characterized by bone disease leading to incomplete bone lengthening and increased risk of fracture with different degrees of severity. In most affected patients, it is caused by the lack, or abnormal synthesis, of type I collagen. The main treatment is with anti-resorptive bisphosphonates to increase bone density, but this fails to address the underlying cause. MSCs may be useful for the treatment of patients with OI because they can differentiate into osteoblasts and supply normal collagen I. MSCs with successfully targeted deletion of mutant collagen will process and form collagen and collagen fibrils similar to wild type cells. In vivo, these cells are able to form ectopic bone in mice.

Cardiovascular diseases: In the last decade, there has been an escalation of studies involving MSCs and cardiovascular disease. Specifically in myocardial infarctions, the plasticity, homing and inflammatory modulation of MSCs are all important properties that come into play following the hypoxia-induced damage to cardiomyocytes due to occluded arteries. The myocardium lacks the ability to efficiently replace these cells, and thus the goal of any treatment is the replacement of cardiomyocytes. The use of genetically-modified MSCs attempts to improve the homing, survival and paracrine-mediated effects of MSCs. In some embodiments, the devices and methods described herein for delivery of molecules 110 to MSCs in bone marrow is used to overexpress therapeutic genes in MSCs that will then travel to the heart.

Pulmonary hypertension prevention: Genetically-modified MSCs have been used to prevent lung ischemia by preventing pulmonary hypertension. The devices and methods described herein may be used for delivery of molecules 110 to MSCs in bone marrow.

Cancer: MSCs are capable of preferentially homing to sites of primary and metastatic tumor growth and delivering anti-tumor agents to highly specific niches surrounding the tumors. The two main categories for gene targeting are cytotoxic, or pro-apoptotic genes, and immunostimulatory genes. Receptors for the pro-apoptotic gene TNF-related apoptosis inducing ligand are expressed on many types of tumors and when soluble forms of the ligand are expressed in MSCs, and properly localized in xenograft mouse models of human cervical and breast cancers and gliomas, have caused the decrease in proliferation and tumor size and increased apoptosis and survival time. Inducible nitric oxide synthase has also been shown to be potentially powerful anti-tumor therapy that when delivered to a fibrosarcoma model in mice by genetically-modified MSCs reduced tumor growth. The second class of therapeutic transgenes is immunomodulatory targets. MSCs engineered to express IL-2, -7, -12 and -18 have decreased tumor size in rodent xenograft models of primary, established and metastatic tumors. MSCs bearing other immune cytokines such as IFN-α and -β and CX3CL1/fractalkine increase tumor cell apoptosis and animal survival times in prostate, lung, pancreatic and skin cancers through activation of innate immune activity such as natural killer cells, or adaptive immune response through increased activation of T cells.

Type I Diabetes: Type 1 diabetes (T1D) results from an organ-specific autoimmune-mediated loss of insulin-secreting β cells in the pancreas. People with T1D manage their blood glucose levels using exogenous insulin therapy; however, this does not eliminate the development of long-term diabetic complications such as retinopathy, nephropathy, and neuropathy. Currently, pancreas or islet transplantation remains the only cure; however, these treatments are limited by a shortage of donor organs and the requirement for life-long immunosuppression. mesenchymal stem cells (MSCs) are an attractive alternative target cell for the autologous and allogeneic treatment of T1D

Targeting of the central nervous system: Mesenchymal stem cells (MSCs), for its homing properties, can travel to various site of the body and perform the repair from the intravenous line to the CNS, acting as vehicles, and it has been shown that they do not arouse meaningful immune responses.

In the event of inconsistent usages between this document and any documents so incorporated by reference, the usage in this document controls.

Delivery to the Eye

A. Background

The retina is a thin layer of tissue that lines the back of the eye. The purpose of the retina is to receive light that the lens has focused, convert the light into neural signals, and send these signals on to the brain for visual recognition. Thus, the retina is central to the process of vision wherein light is converted to signals that can be interpreted in the brain.

FIG. 28 illustrates a cross-sectional portion of a retina R near an optic nerve ON. The central area of the retina, near the optic nerve ON, is called the macula and contains a high density of color-sensitive photoreceptor (light-sensing) cells. These cells, called cones, produce the sharpest visual images and are responsible for central and color vision. The peripheral area of the retina, which surrounds the macula, contains photoreceptor cells called rods, which respond to lower light levels but are not color sensitive. The rods are responsible for peripheral vision and night vision. Both cones and rods are found within a Layer of Rods and Cones (Jacob's membrane) LRC. The optic nerve ON carries signals generated by the photoreceptors (cones and rods). Each photoreceptor is joined to the optic nerve by a tiny nerve branch. The optic nerve is connected to nerve cells that carry signals to the vision center of the brain, where they are interpreted as visual images.

The optic nerve ON and the retina R have a rich supply of blood vessels that carry blood and oxygen. Part of this supply of blood vessels comes from the choroid CH, which is the layer of blood vessels that lies between the retina R and the outer white layer of the eye called the sclera SC. The central retinal artery (the other major source of blood to the retina) reaches the retina R near the optic nerve ON and then branches out within the retina R. Blood drains from the retina R into branches of the central retinal vein. The central retinal vein exits the eye within the optic nerve ON.

A variety of diseases and conditions can lead to dysfunction of the retina and eventual blindness. Inherited retinal degenerations (IRDs), also known as inherited retinal dystrophy, represent a diverse group of progressive, visually debilitating diseases that can lead to blindness. In IRDs, mutations in genes that are critical to retinal function lead to progressive photoreceptor cell death and associated vision loss. In most people, IRDs only affect the eyes. However, some types of IRDs are linked with other health issues. IRDs are genetically heterogeneous, with over 260 disease genes identified to date. IRDs are the leading cause of blindness in working age adults. Altogether, they affect around 1 in 4000 people or over 2 million people worldwide.

The development of treatments and cures to modify or halt the rate of disease progression has been limited to date. Therapies that may slow photoreceptor degeneration due to a range of genetic causes have been investigated. Vitamin A and docosahexaenoic acid have been demonstrated to provide modest reductions in the rate of disease progression in patients with retinitis pigmentosa (RP). Oral valproic acid was reported to slow visual field progression in a case series of RP patients, but a randomized clinical trial of valproic acid treatment in patients with autosomal dominant RP showed no significant difference between patients treated with valproic acid and placebo.

Advances in high-throughput screening have accelerated the pace of identifying cellular targets and candidate neuroprotective agents. Oxidative damage has been implicated in photoreceptor degeneration, and N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) have been shown to prevent retinal degeneration in preclinical studies of RP. Nonspecific neurotrophic factor therapy with ciliary neurotrophic factor (CNTF) has been shown to slow photoreceptor degeneration in a number of animal models, but did not demonstrate visual function benefit in human clinical trials of patients with early or advanced RP.

In addition, significant effort has been directed toward developing gene augmentation therapies for specific genetic forms of IRD. Successful clinical trials of gene augmentation therapy for RPE65- and CHM-associated retinal degeneration have been reported. Genome editing and genetically directed pharmacologic therapies, including antisense oligonucleotides, premature termination codon read-through strategies, base editing, and RNA editing may also be promising approaches for genetic forms of disease that may be not amenable to gene augmentation therapies.

However, introduction of genetic material into cells has its challenges, as mentioned previously. Unlike many other biological molecules and therapeutic compounds, DNA sequences and nucleic acid are not designed to travel across cell membranes. Further, they lack endocytotic factors that would induce alternate mechanisms of transport into cells. Therefore, all gene therapies require a combination of at least two features: the therapeutic encoded molecule (DNA sequence, gene, siRNA, etc) and a route of administration into the targeted cells. One way that genetic material has been delivered to cells is with viral vectors. Various drawbacks of viral vectors have been mentioned previously. In addition,

Electroporation has been known for over three decades as a method to disrupt cell membrane integrity and result in macromolecule uptake for cells in suspension. A large number of in vitro investigations to test the effects of genes on cell morphology and behavior rely on electroporation as their steadfast method of delivery into the cells. Generally, cells are suspended in media that is pipetted into a cuvette. The cuvette is comprised of two plate electrodes at an established distance apart. A sequence of pulsed electric fields is then delivered to the cells to transfect the genetic material. The vitro environment permits very precise control of transfection conditions, permitting high levels of gene delivery without killing too large of a proportion of the cells. Oftentimes, protocols have been developed that are optimized specifically for different cell lines. Overall, transfection in vitro has a high reliability for generating fundamental understanding of gene effects or producing transgenic experimental animal models. However, it has difficulty in implementing viable clinical gene therapies. The need for transfecting cells in culture requires several additional steps in translating to a therapy for patients. Most often, an apheresis-based approach is used to deliver a gene therapy. This involves sampling a patient's cells through a blood draw or biopsy. The targeted cell population are then isolated and concentrated. Oftentimes, the cell population is also amplified to produce a meaningful population size of transfected cells. Once the cells have been prepared, they are then electroporated with the genetic therapeutic molecule. Following transfection, the cells are then redistributed at the necessary site in the patient, such as the blood or the target location in the targeted organ. These steps add substantial burden, cost, invasiveness, time, and risk to delivering a gene therapy. Overall, these numerous and substantial drawbacks have prevented widespread adoption of pulsed electric field-based gene therapies for most potential therapeutic options.

B. Overview

Devices, systems and methods are provided for delivering molecules, particularly small molecules and/or macromolecules, to cells within the body, particularly to target cells within the eye, more particularly to target cells within the retina. Example molecules include plasmids, RNAs (e.g. messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA), antisense oligonucleotides, proteins and/or materials which invoke genetic or epigenetic changes in the cellular behavior, to name a few. Such molecules are typically beneficial in the treatment of a variety of diseases and conditions, such as inherited retinal degenerations (IRDs) including but not limited to Retinitis pigmentosa (RP), Rod dystrophy or rod-cone dystrophy, Usher syndrome (USH), Bietti crystalline dystrophy (BCD), Batten disease, Bardet-Biedl syndrome (BBS), Alport syndrome, Leber congenital amaurosis (LCA) or early onset retinal dystrophy (EORD), Cone dystrophy, Cone-rod dystrophy (CORD), Achromatopsia, Congenital stationary night blindness (CSNB), Macula dystrophy, Stargardt's disease, Best disease, Pattern dystrophy, Sorsby fundus dystrophy, Doyne's honeycomb dystrophy, Choroideremia, X-linked retinoschisis (XLRS), etc.

The molecules may be delivered to the eye by a variety of mechanisms, including injection into a vitreous space (or the area between the lens and the retina), injection in a suprachoroidal space (a potential space between the sclera and choroid that traverses the circumference of the posterior segment of the eye) and/or by injection into a subretinal bleb (a small area created by separating the Layer of Rods and Cones (LRC) from its supporting retinal pigment epithelium (RPE)). The molecules are then delivered through the cell wall of the target cells with the use of pulsed electric fields so that the molecules are delivered thereto and able to carry out the desired effect. Thus, in the case of gene therapy, the cells become transfected with the nucleic acids delivered by the molecules, without the use of viruses.

These devices, systems and methods are superior to delivery by adeno associated virus (AAV), a known virus-based gene therapy. AAV has a limited payload capacity; AAV can only accommodate genes<4.7 kb and has limited space for genetic control elements (e.g. promoters, etc). There are more than 300 disease genes that are too large for AAV-based gene therapies. These include genes leading to many relatively common diseases, such as Stargardt disease, Usher 1B and 1D, and Leber congenital amaurosis-10 (LCA10). In contrast, the non-viral delivery described herein can accommodate genes>10 kb and is not limited in space. Thus, many of the genes precluded from use with AAV delivery can be delivered by the devices, systems and methods described herein.

The devices, systems and methods deliver pulsed electric field energy to the cells with an energy delivery system 100. FIG. 29 illustrates an embodiment of an energy delivery system 100 configured for delivery of energy to portions of the eye. In this embodiment, the energy delivery system 100 comprises a specialized energy delivery device 102 removably connectable to a waveform generator 104. In this embodiment, the generator 104 includes a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage/retrieval unit 156 (such as a memory and/or database), and an energy-storage sub-system 158 which generates and stores the energy to be delivered. Additional accessories and equipment may be utilized. The energy delivery device 102 delivers energy provided by the waveform generator 104 according to the one or more energy delivery algorithms 152.

In this embodiment, the energy delivery device 102 comprises a rigid shaft 106, an electrode body 108 and a handle 105. Typically, the electrode body 108 comprises one or more electrodes. The shaft 106 is sufficiently rigid so as to allow passage through a surface of the eye so that its distal end enters an interior portion of the eye. In this embodiment, the shaft 106 has an interior lumen configured for the passage of the electrode body 108 therethrough so that at least a portion of the electrode body 108 extends beyond the distal end of the shaft 106. In this embodiment, the electrode body 108 has the form of a conductive rod or wire which acts as an electrode and which is sized and configured to pass through the lumen in the shaft 106. In this embodiment, the handle 105 includes an actuator 132 that is manipulatable so as to manipulate the electrode body 108. For example, in this embodiment, the actuator 132 comprises a button which advances and retracts the electrode body 108 from the distal end of the shaft 106. This allows the electrode body 108 to be retracted while the shaft 106 is inserted into the eye and the electrode body 108 to be advanced toward or into the target tissue area once the shaft 106 is desirably positioned. It may be appreciated that other types of actuators 132 may be used including slides, ratchets, knobs, dials, sensors, etc. Once the electrode body 108 is exposed in a desired location, pulsed electric field energy is passed from the generator 104 to the target tissue area via the electrode body 108. In this embodiment, the shaft 106 is non-conductive or insulated so that the energy provided by the electrode body 108 emanates from the exposed portion of the electrode body 108.

It may be appreciated that many of the energy delivery devices 102 described herein throughout are configured to provide monopolar energy delivery. In such instances, a return electrode is positioned on the surface of the patient, such as on the torso, buttock or lower limb. When treating the eye, the return electrode may be positioned closer to the eye, such as on the surface of the eye. It may also be appreciated that in some embodiments the energy delivery devices 102 are configured to provide energy in bipolar or multi-polar arrangements. In such embodiments, the energy delivery device 102 includes more than one electrode body 108 which together act in a bipolar or multi-polar arrangement, or the electrode body is configured to function in a bipolar or multi-polar manner, such as with more than one electrode or portions of the electrode body 108 acting as a bipolar pair or multi-polar set. Likewise, in some embodiments, a portion of the bipolar pair or multi-polar set is located on a separate device. Consequently, separate devices that deliver or receive electric current will be described as energy delivery devices 102 even if they are used to receive electric current. Likewise, energy delivery body 108 and electrode body 108 are used interchangeably. This is to simplify the nomenclature since the functionality of delivering or receiving may vary with changes in polarities of a bipolar pair or multi-polar set. It may be appreciated that the energy delivery bodies and the electrode bodies may include one or more electrodes.

It may be appreciated that, in some embodiments, the energy delivery device 102 also delivers the molecules that are to be taken up by the target cells. However, in other embodiments, the molecules are delivered by a separate device, such as by needle injection. Optionally, the molecules may be delivered both by the energy delivery device and by a separate device.

The energy, such as pulsed electric fields (PEFs) energy, is provided by the generator 104 and delivered to the tissue within the eye via at least one electrode body 108. These electric pulses are provided by at least one energy delivery algorithm 152. In some embodiments, each energy delivery algorithm 152 prescribes a signal having a waveform comprising a series of pulses. The algorithm 152 specifies parameters of the signal such as energy amplitude (e.g. voltage) and duration of applied energy, which is comprised of the number of pulses, the pulse widths and the delay between pulses, to name a few. In some embodiments, one or more of the electrode bodies are small and tend to dissipate large amount of energy around the electrode. Therefore, an optimal delivery of energy is desired. In some embodiments, a large DC-link capacitance with half transistor bridges is utilized to deliver efficient delivery pulses in such instances. In some instances, this is preferred in relation to pulse voltages delivered by power amplifiers (limited bandwidth) or exponential decay generators. In some embodiments, a feedback loop based on sensor information and an auto-shutoff specification, and/or the like, may be included.

In some embodiments, biphasic pulses may be used. In such embodiments, additional parameters may include switch time between polarities in biphasic pulses and dead time between biphasic cycles. In some instances, biphasic waveforms are convenient to reduce muscle stimulation in patients. This is particularly important in the application where slight movement of the electrode body can easily result a non-effective therapy. Biphasic waveforms involve rapid change of phases/polarities of the signal to minimize nerve activation during transition between polarity. Multiple fast switching elements (e.g. MOSFET, IGBT Transistors) are desired and are employed and configured in, for example, H-bridge structure or full bridge.

C. Subretinal Bleb

As mentioned previously, molecules may be delivered to the eye by a variety of mechanisms, including injection into the vitreous space (or the area between the lens and the retina), injection in a suprachoroidal space (a potential space between the sclera and choroid that traverses the circumference of the posterior segment of the eye) and/or by injection into a subretinal bleb (a small area created by separating the Layer of Rods and Cones (LRC) from its supporting retinal pigment epithelium (RPE)). FIG. 30 illustrates an embodiment of a procedure for creating a subretinal bleb RB that may be described as follows.

Prior to surgery, the pupil of the eye E is dilated and topical antibiotic drops are applied. The surgery is typically performed under general anesthesia supplemented by local anesthetic.

In this embodiment, the subretinal bleb is described as created with the use of a subretinal injection apparatus 201. Such an apparatus 201 is configured for delivery of a solution, such as a solution of the molecules, to create the bleb. In such embodiments, the energy is provided by an energy delivery device 102 which is a separate device. However, it may be appreciated that the procedure may be undertaken with an energy delivery device 102 that is equipped with the ability to deliver a solution, such as a solution of the molecules. In such instances, the bleb is created with the energy delivery device 102 and the energy is delivered with the same device.

In this embodiment, a subretinal injection apparatus 201 is prepared on the sterile surgical field. The injection apparatus 201 and any extension tubing are secured to a syringe (not shown) containing the molecules (e.g. plasmids, RNAs, oligonucleotides, deoxyoligonucleotides, antisense oligonucleotides, or proteins, etc.) The volume of molecules available for injection is confirmed.

A lid speculum is placed and a standard 3-port pars plana vitrectomy is performed. The tip of an infusion cannula is directly visualized through the pupil to confirm its intravitreal position. At this point, the infusion should remain running until the completion of the operation.

Core vitrectomy is performed at high cutting rate and low suction settings. The vitreous is removed as completely as possible. After completion of the core vitrectomy, a complete posterior vitreous detachment (PVD) is confirmed. At this point, the vitreous cortex is no longer attached to the macular area. The remaining mobilized vitreous is then removed as completely as possible with the vitreous cutting instrument.

Prior to subretinal injection of the molecules, the retina is inspected. Any retinal breaks identified are treated. The injection of molecules is performed in two steps. First, the apparatus tip is positioned so as to indent the retina and drape the retina over the tip. The location should be far enough away from the fovea to minimize mechanical stress to this structure caused by the injection. A small amount of molecules is injected to confirm that the tip is not occluded and it is properly positioned. Next, if a bleb is raised, the molecules are injected to deliver a total volume up to 0.3 mL or any intended volume (e.g. up to 1 mL). If no bleb is created during the test injection, or if further injection does not increase size of the bleb, the cannula tip is repositioned distant from the original retinotomy, and the sequence is repeated. The second retinotomy should be far enough away that any additional injected material does not connect with and reflux through the first retinotomy site. After the full contents of the syringe have been discharged, the injection apparatus 201 is held in place (to allowing the remaining contents of the syringe to be delivered). After this time, the injection apparatus 201 is removed.

The subretinal bleb RB provides a space within which to place molecules adjacent the retinal pigment epithelial cells (RPE) and the photoreceptors of the Layer of Rods and Cones (LRC) and/or an electrode body 108 for delivering PEF energy. In some embodiments, this proximity increases the ability of the cells to uptake the molecules upon delivery of the pulsed electric field energy. In some embodiments, aspects of the pulsed electric field energy determine which cells uptake the molecules and the amount of uptake. Uptake may be controlled or influenced by a variety of factors such as the choice of signal parameters, signal waveforms, electrode polarity, electrode placement, type of molecules, impedance characteristics of the anatomical environment, etc. Thus, in some embodiments, the photoreceptors of the LRC uptake the molecules. And, in other embodiments, the RPE uptake the molecules, and in still other embodiments, both the photoreceptors and the RPE uptake the molecules. Likewise, in other embodiments, other retinal layers, tissues of the eye or surrounding areas may be targeted. It may be appreciated that in some embodiments, uptake is selective based on the signal parameters, such as wherein one set of parameters causes uptake by one portion of the anatomy (e.g. LRC), one set of parameters causes uptake by another portion of the anatomy (e.g. RPE) and yet another set of parameters causes uptake by two or more portions of the anatomy (e.g. LRC and RPE). Likewise, changing reversing or changing polarity of the electrode bodies can also change which cells uptake the molecules.

FIG. 31 illustrates positioning of an embodiment of an energy delivery device 102 into the eye E so that the electrode body 108 is positioned within the subretinal bleb RB. In this embodiment, the shaft 106 passes through the surface of the eye E and the retina R so that the handle 105 resides outside of the eye E. Although the energy delivery device 102 is illustrated as entering the eye E at a different location than the injection apparatus 201, it may be appreciated that, upon removal of the injection apparatus 201, the energy delivery device 102 may be introduced to the eye E through the same opening. Once the energy delivery device 102 is desirably positioned, pulsed electric field energy is delivered through the electrode body 108 from the generator 104. In this example, the electrode body 108 has a positive charge and a return electrode outside of the eye, such as a remote return electrode is positioned on the surface of the patient, has a negative charge. In this embodiment, the electrode body 108 delivers energy to the subretinal bleb RB in a monopolar fashion wherein the energy flows out of the subretinal bleb RB (indicated by arrows), driving negatively molecules 110 within the bleb RB into the retina R and optionally other structures of the eye E. It may be appreciated that the directions that the energy drives the molecules 110 depends on the charge of the molecules 110, the polarity of the electrodes and the location of the electrodes, to name a few. Other combinations and outcomes will be described in later sections. The energy delivery device 102 is then removed.

FIGS. 32A-32B illustrate molecules 110 that have entered the RPE upon application of pulsed electric field energy. In particular, FIG. 32A illustrates the eye E wherein a subretinal bleb RB has been created (indicated by an asterick *) containing the molecules. Energy is applied so that cells of the retina are able to uptake the molecules. FIG. 32B illustrates the molecules 110 residing in the RPE.

Prior to closing the infusion sclerotomy, the infusion line is clamped in order to prevent a supero-choroidal infusion. The incisions are sutured closed. Next, subconjunctival injection of 0.5 mL of 4 mg/mL dexamethasone solution (for acute inflammation) and 0.5 mL of antibiotic solution is administered. The ocular surface is dressed with ointment and a patch, and an eye shield is put in position and secured over the eye which received the subretinal injection.

D. Example Delivery Options

It may be appreciated that a variety of delivery options may be used, such as various locations of molecule delivery, various locations of electrode placement, various types of electrode arrangements, various types of waveforms and various combinations of signal parameters, to name a few. At least some combinations will be described herein but are not so limited.

Table 8 sets forth a variety of combinations of example delivery options. Here, molecules 110 are delivered to a variety of locations, such as to a subretinal bleb, the vitreous space, or a combination of a subretinal bleb and vitreous space. Likewise, the electrode body 108 may be positioned in a variety of locations, such as in the subretinal bleb, vitreous space, against the cornea or in a periocular space (e.g. subconjunctival, sub-Tenon, or retrobulbar). It may be appreciated that, in some instances, the subretinal bleb may be exchanged for the suprachoroidal space in these combinations when so desired. Likewise, the suprachoroidal space may be utilized in combination with one or more blebs for molecule and/or energy delivery. Injections of 10-50 μL into the suprachoroidal space have been demonstrated to be well tolerated with low risk of ocular complications, and in some instances up to 1 mL may be injected. Suprachoroidal delivery may be particularly useful when targeting the retinal pigment epithelium.

The suprachoroidal space has been long known as a potential space between the choroid and the sclera. While the inner border of the choroid, which is the Bruch's membrane, is compact, the outer border is more a zone of transition, consisting of several fibrous lamellae with variable thickness. The suprachoroidal space has been shown to be present in approximately 50% of people above the age of 50 years. The presence of the suprachoroidal space has been correlated to hyperopic refractive error, and is generally absent in the eyes of young, healthy persons. It has been theorized that in hyperopic eyes, there is increased hydrostatic pressure from compression of the vortex veins by the sclera, resulting in a subclinical suprachoroidal effusion that makes the small amount of fluid in the suprachoroidal space visible on imaging. It has also been suggested that with age there is an increase in leakage of proteins from choroidal vessels to the suprachoroidal space, increasing its osmotic pressure as well, and results in the higher rates of its detection in older individuals. The suprachoroidal space is also present in a variety of patients with particular conditions. Therefore, in some instances, the suprachoroidal space may be utilized as a location for delivery.

TABLE 8 Molecule delivery location Electrode body location Type Bleb only Bleb monopolar Bleb only Vitreous space monopolar Bleb only Bleb & Vitreous space bipolar Bleb only Bleb & Cornea bipolar Bleb only Bleb & Periocular bipolar Bleb only Bleb & Suprachoroidal bipolar Bleb only Vitreous space & Cornea bipolar Bleb only Vitreous space & bipolar Periocular Bleb only Vitreous space & bipolar Suprachoroidal space Bleb only Cornea & Periocular bipolar Bleb only Cornea & Suprachoroidal bipolar space Bleb only Suprachoroidal space & bipolar Periocular Vitreous space only Bleb monopolar Vitreous space only Vitreous space monopolar Vitreous space only Bleb & Vitreous space bipolar Vitreous space only Bleb & Cornea bipolar Vitreous space only Bleb & Periocular bipolar Vitreous space only Bleb & Suprachoroidal bipolar space Vitreous space only Vitreous space & Cornea bipolar Vitreous space only Vitreous space & bipolar Periocular Vitreous space only Vitreous space & bipolar Suprachoroidal Vitreous space only Cornea & Periocular bipolar Vitreous space only Cornea & Suprachoroidal bipolar space Vitreous space only Suprachoroidal space & bipolar Periocular Bleb & Vitreous space Bleb monopolar Bleb & Vitreous space Vitreous space monopolar Bleb & Vitreous space Bleb & Vitreous space bipolar Bleb & Vitreous space Bleb & Cornea bipolar Bleb & Vitreous space Bleb & Periocular bipolar Bleb & Vitreous space Vitreous space & Cornea bipolar Bleb & Vitreous space Vitreous space & bipolar Periocular Bleb & Vitreous space Vitreous space & bipolar Suprachoroidal Bleb & Vitreous space Cornea & Periocular bipolar Bleb & Vitreous space Cornea & Suprachoroidal bipolar space Bleb & Vitreous space Suprachoroidal space & bipolar Periocular Suprachoroidal space only Bleb monopolar Suprachoroidal space only Vitreous space monopolar Suprachoroidal space only Suprachoroidal space monopolar Suprachoroidal space only Bleb & Vitreous space bipolar Suprachoroidal space only Bleb & Cornea bipolar Suprachoroidal space only Bleb & Periocular bipolar Suprachoroidal space only Bleb & Suprachoroidal bipolar space Suprachoroidal space only Vitreous space & Cornea bipolar Suprachoroidal space only Vitreous space & bipolar Periocular Suprachoroidal space only Vitreous space & bipolar Suprachoroidal Suprachoroidal space only Cornea & Periocular bipolar Suprachoroidal space only Cornea & Suprachoroidal bipolar space Suprachoroidal space only Suprachoroidal space & bipolar Periocular Suprachoroidal space & Vitreous space monopolar Bleb/Vitreous space

A variety of these combinations are described and illustrated in more detail herein. It may be appreciated that a multitude of combinations exist and only a sampling are provided herein. Likewise, more than one subretinal bleb may be created in a single eye. Therefore, molecules and/or energy delivery devices may be delivered to more than one bleb for further combinations. Even the combinations listed in Table 1 include subcombinations, such as having two variations of charge (positive and negative pole) for each bipolar pair. Likewise, location of the molecules 110 and/or location of the electrode body 108 within the anatomical location can also impact the outcome. For example, positioning molecules within one area of the vitreous space may have a different outcome than molecules positioned within a different area of the vitreous space. Or, molecules 110 concentrated in one area of the vitreous space may have a different outcome than molecules diffused throughout the vitreous space. Likewise, anatomical features may impact the outcome, such the natural impedance of particular tissues that may influence the movement of molecules 110. In addition, different types of waveforms may cause the molecules 110 to move differently, including moving at different speeds and trajectories. For example, some waveforms induce translation while others induce rotations, jiggling or combinations of these with translation, to name a few. Thus, it may be appreciated that the examples provided herein are a sampling and are not considered to be limiting.

FIG. 33 illustrates an embodiment wherein the molecules 110 are delivered to the subretinal bleb RB and the electrode body 108 is positioned within the subretinal bleb RB for monopolar energy delivery thereto. Here, the molecules are negatively charged, the electrode body 108 is negatively charged and the remote return electrode is positively charged. This typically causes the molecules to be driven away from the electrode body 108, such as into the retinal pigment epithelium RPE and Layer of Rods and Cones LRC. In some instances, steering toward the RPE or toward the LRC may be achieved with placement of the electrode body 108 within the subretinal bleb RB, choice of waveform or other factors. In similar embodiments, molecules 110 are also added to the vitreous space V. It may be appreciated that this area between the lens and the retina is referred to as the vitreous space V herein regardless of whether this area is filled with vitreous humor or another solution. Molecules 110 in the vitreous space 110 are typically driven into portions of the retina more distant from the subretinal bleb RB. It may be appreciated that the molecules 110 may be specifically positioned in the vitreous space V to predispose movement of the molecules 110 into particular areas of the retina.

FIG. 34 illustrates an embodiment wherein the molecules 110 are delivered to the subretinal bleb RB and the electrode body 108 is positioned within the vitreous space V of the eye E for monopolar delivery thereto. Again, here the molecules are negatively charged, the electrode body 108 is negatively charged and the remote return electrode is positively charged. This typically causes the molecules 110 to be driven away from the electrode body 108, such as into the retinal pigment epithelium RPE. It may be appreciated that, depending on the electrode orientation and the polarity, the molecules 110 may be pushed towards and into the retinal pigment epithelial RPE cells. In some embodiments, changing polarity of pulses (biphasic) every pulse or after multiple pulses (e.g. 4 up, 4 down . . . 2 up, 2 down, etc.) drives the molecules 110 in one direction and then another which may allow for targeting of both the retinal pigment epithelial RPE cells and the Layer of Rods and Cones LRC. In similar embodiments, molecules 110 are also added to the vitreous space V. In such embodiments, molecules 110 in the vitreous space 110 are typically driven into the LRC. It may be appreciated that the molecules 110 may be specifically positioned in the vitreous space V to predispose movement of the molecules 110 into particular areas of the retina. It may be appreciated that in some embodiments, different types of molecules 110 are placed in the bleb and the vitreous space.

FIG. 35 illustrates an embodiment wherein the molecules 110 are delivered to the vitreous space V and the electrode body 108 is also positioned in the vitreous space V for monopolar delivery thereto. In such instances, a subretinal bleb RB is not created since the desired access is directly to the vitreous space V. Again, here the molecules 110 are negatively charged, the electrode body 108 is negatively charged and the remote return electrode is positively charged. This typically causes the molecules to be driven away from the electrode body 108, such as into the LRC. It may be appreciated that in some instances the molecules 110 are concentrated in a specific region or area of the vitreous space V prior to delivery of the energy. For example, in some embodiments, molecules 110 are delivered to a specific location within the vitreous space V, such as to be close to a target tissue area and/or to create a particular concentration to provide enhanced and/or more predictable delivery of molecules 110 to the target cells. In such instances, the electrode body 108 may be desirably placed within the vitreous space V in relation to the concentration of molecules 110 to optimize delivery. In some embodiments, this is within the same location in the vitreous space V and, in other embodiments, this is adjacent to such location so as to steer the molecules 110 in a particular direction. For example, in some embodiments, the molecules 110 are positioned adjacent the LRC and the electrode body 108 is positioned adjacent the molecules 110 so that the electric current of the energy drives the molecules 110 away from the electrode body 108, into the LRC.

In other embodiments, energy is delivered in a bipolar manner, such as with the use of two separate energy delivery devices 102 or with the use of a single energy delivery device 102 having a pair or more of electrode bodies. For example, FIG. 36 illustrates the molecules 110 disposed within the subretinal bleb RB and two electrode bodies positioned in the eye E to deliver the energy from one to another in a bipolar manner. Referring to FIG. 36 , the molecules 110 are shown within the subretinal bleb RB and a first energy delivery device 102′ having a first electrode body 108′ is shown inserted into the eye E so that the first electrode body 108′ is disposed within the subretinal bleb RB. In this embodiment, a second energy delivery device 102″ having a second electrode body 108″ is shown inserted into the eye E so that the second electrode body 108″ is disposed within the vitreous space V of the eye E. In this embodiment, electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. With both bipolar electrodes in the eye in this arrangement, negatively charged molecules (e.g. DNA) may be pushed, for example, towards and into the Layer of Rods and Cones LRC. It may be appreciated that the location of the second electrode body 108″ in the vitreous space V may be chosen to elicit particular effects, such as driving the molecules 110 into a particular portion of the LRC or increasing the magnitude of the drive. In a similar embodiment, the second electrode body 108″ is positioned on the cornea C rather than in the vitreous space V. The results may be similar to those described in relation to FIG. 36 , however the energy field may cause a small increase in delivery to the RPE however the majority of the molecules 110 will continue to be driven into the LRC.

FIG. 37 illustrates an embodiment similar to that of FIG. 36 wherein two electrode bodies are utilized, one in the subretinal bleb RB and one in the vitreous space V. However, here the molecules 110 are disposed in the vitreous space V and here the polarities of the electrode bodies are switched. Thus, the first electrode body 108′ is positioned within the vitreous space V and the second energy delivery device 102″ having a second electrode body 108″ is shown inserted into the eye E so that the second electrode body 108″ is disposed within the subretinal bleb RB. Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. Again, here the molecules are negatively charged, the first electrode body 108′ is negatively charged and the second electrode body 108″ is positively charged. This typically causes the molecules to be driven away from the first electrode body 108′, such as into the LRC. Again, it may be appreciated that the location of the electrode bodies 108′, 108″ and the molecules 110 within the vitreous space V may be chosen to elicit particular effects, such as driving the molecules 110 into a particular portion of the LRC or increasing the magnitude of the drive. In a similar embodiment, the first electrode body 108′ is on the cornea C. The results may be similar to those described in relation to FIG. 37 .

FIG. 38 illustrates a similar embodiment to FIG. 37 , however molecules 110 are disposed in both the subretinal bleb RB and the vitreous space V. Again, the first electrode body 108′ is positioned within the vitreous space V and the second electrode body 108″ is disposed within the subretinal bleb RB. Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. In some embodiments, the molecules are negatively charged, the first electrode body 108′ is negatively charged and the second electrode body 108″ is positively charged. This typically causes the molecules 110 to be driven away from the first electrode body 108. Thus, molecules 110 in the vitreous space V are driven into the LRC and molecules in the subretinal bleb RB are driven away from the electrode body 108′, such as in to the LRC or RPE. It may be appreciated that in some embodiments, different types of molecules 110 are placed in the bleb and the vitreous space.

FIG. 39 also illustrates bipolar energy delivery to the eye. Here, a first energy delivery device 102′ having a first electrode body 108′ is shown inserted into the eye E so that the first electrode body 108′ is disposed within the vitreous space V. In this embodiment, a second energy delivery device 102″ having a second electrode body 108″ is shown inserted into a retrobulbar space RBS of the eye E. The retrobulbar space RBS is behind the eye E, near the optic nerve ON within the muscle cone MC. Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. In this embodiment, the molecules 110 are disposed within a subretinal bleb RB. With the devices 102′, 102″ in this arrangement, negatively charged molecules (e.g. DNA) are typically pushed towards and into the RPE. It may be appreciated that if the polarity of the electrode bodies 108′, 108″ were switched, negatively charged molecules may be pushed towards and into the LRC. In a similar embodiment, the first electrode body 108′ is on the cornea C while the second electrode body 108″ is positioned in a retrobulbar space RBS. The results may be similar to those described in relation to FIG. 39 , however in some instances the energy field is more homogenized. This may lead to a more consistent transfer of molecules 110 to the RPE.

Referring to FIG. 40 , the molecules 110 are again shown within the subretinal bleb RB, however here the first energy delivery device 102′ having a first electrode body 108′ is shown inserted into the eye E so that the first electrode body 108′ is disposed within the subretinal bleb RB. In this embodiment, the second energy delivery device 102″ having a second electrode body 108″ is shown inserted into a retrobulbar space RBS of the eye E. Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. With the devices 102′, 102″ in this arrangement, negatively charged molecules (e.g. DNA) may be pushed, for example, towards and into the RPE, however a small amount may be pushed toward and into the LRC.

FIG. 41 illustrates a similar embodiment, however here the molecules 110 are disposed within the vitreous space V rather than the subretinal bleb RB. In this embodiment, energy is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. With the devices 102′, 102″ in this arrangement, negatively charged molecules (e.g. DNA) are repelled by the first electrode body 108′ but follow the electric field around the first electrode body 108′ toward the second electrode body 108″. Thus, the molecules 110 may be driven towards the sides of the retina, rather than directly into the portion of the retina overlaying the subretinal bleb RB. This may be beneficial when desiring treatment of specific portions of the retina in these areas.

FIG. 42 illustrates a similar embodiment to FIGS. 40-41 , however here the molecules 110 are disposed in both the subretinal bleb RB and the vitreous space V. Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. In some embodiments, the molecules are negatively charged, the first electrode body 108′ is negatively charged and the second electrode body 108″ is positively charged. This typically causes the molecules 110 to be driven away from the first electrode body 108. Thus, molecules 110 in the vitreous space V are driven into the LRC and molecules in the subretinal bleb RB are driven into the RPE. It may be appreciated that in some embodiments, different types of molecules 110 are placed in the bleb and the vitreous space.

FIG. 43 illustrates an embodiment wherein the molecules 110 are delivered to the vitreous space V and a first electrode body 108′ is positioned within the vitreous space V. In such instances, a subretinal bleb RB is not created since the desired access is directly to the vitreous space V. In this embodiment, a second electrode body 108″ is positioned against the cornea C. It may be appreciated that a variety of different designs may be utilized to position an electrode body against a cornea C. A variety of scleral type contact lens electrodes may be utilized. Examples include a Burian-Allen contact lens electrode and a DTL™ (Diagnosys LLC) fiber electrode which are the most frequently used electrodes in the US for recording the electroretinogram (ERG) and can be used to deliver the electric currents described herein. Other examples include a Gold Ring Electrode and a Jet Contact Lens Electrode. Other types of devices include tweezer-trodes used around the eye or a speculum.

Electric current is delivered to the first electrode body 108′ and flows to the second electrode body 108″ in a bipolar fashion. Again, here the molecules are negatively charged, the first electrode body 108′ is negatively charged and the second electrode body 108″ is positively charged. Thus, molecules 110 on disposed between the first electrode body 108′ and the LRC may be driven away from the first electrode body 108′ toward the LRC, following the electric field which then bends around toward the second electrode body 108″. Thus, these molecules 110 are driven toward and into the LRC. It may be appreciated that positioning the molecules 110 near the LRC and the first electrode body 108′ between the molecules 110 and the second electrode body 108″ may accentuate this effect. It may be appreciated that in a similar embodiment the second electrode body 108″ is positioned in a retrobulbar space RBS rather than on the cornea C. With this modification, the molecules 110 are also driven into the LRC, possibly with stronger drive depending on the circumstances.

E. Example Devices

The energy may be delivered by a variety of energy delivery devices 102. Typically, the energy delivery device 102 comprises an elongate shaft 106 having a distal end, capable of being advanced to the target tissue with the body, and an electrode body 108 disposed near the distal end. The electrode body 108 comprises one or more electrodes that delivers the PEF energy to the target tissue. Such devices are configured for accessing target tissue on or within the eye E. In some embodiments, such devices are configured for accessing target tissue within or near the retina R.

As mentioned previously, in some embodiments, the energy delivery device 102 is configured to deliver a solution, such as containing molecules 110, to the target tissue. FIGS. 44A-44B illustrate such an embodiment. FIG. 44A illustrates an embodiment of an energy delivery device 102 comprises a rigid shaft 106, an electrode body 108 and a handle 105. The shaft 106 is sufficiently rigid so as to allow passage through a surface of the eye so that its distal end enters an interior portion of the eye. In this embodiment, the shaft 106 has an interior lumen configured for the passage of the electrode body 108 therethrough so that at least a portion of the electrode body 108 extends beyond the distal end of the shaft 106. In this embodiment, the energy delivery device 102 includes an injection port 300 which fluidly connects to the interior lumen of the shaft 106. In some embodiments, the injection port 300 includes a luer fitting 302 for attachment of a syringe. Typically, a syringe containing a desired solution is attached to the luer fitting 302. When delivery of the solution is desired, the electrode body 108 is retracted so as to allow passage of the solution through the interior lumen of the shaft 106. In this embodiment, the handle 105 includes an actuator 132 that is manipulatable so as to manipulate the electrode body 108. In this embodiment, the actuator 132 comprises a slide which advances and retracts the electrode body 108 from the distal end of the shaft 106. This allows the electrode body 108 to be retracted.

The solution is then injected, discharging through the distal end of the shaft 106 to the target tissue area. The electrode body 108 is then advanced through the interior lumen by sliding the actuator 132. It may be appreciated that in some embodiments the interior lumen is sized so as to allow solution to be injected therethrough while the electrode body 108 is in place. This allows simultaneous delivery of solution and energy to the target tissue area.

FIG. 44B provides an additional view of an embodiment of an energy delivery device 102 having an injection port 300 which fluidly connects to the interior lumen of the shaft 106. In this view, a syringe 304 is shown that is attachable to the luer fitting 302. Likewise, a cable 310 is shown extending from the handle 105, wherein the cable 310 has a fitting 312 configured for connection to the generator 104. This provides the electrical energy to the energy delivery device 102, particularly the electrode body 108.

It may be appreciated that the energy delivery devices 102, particularly the electrode bodies 108, may take a variety of forms. FIG. 45 illustrates an embodiment of an energy delivery device 102 showing a close up of its distal end. Here, the energy delivery device 102 comprises a shaft 106 having an interior lumen 107 through which the electrode body 108 extends. In this embodiment, the electrode body 108 comprises a conductive rod or wire 320. In this embodiment, the wire 320 has a pointed distal tip configured to penetrate tissue, however it may be appreciated that the distal tip may have a variety of shapes, including blunt or rounded shapes.

In this document, the terms “a” or “an” are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of “at least one” or “one or more.” In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated. In this document, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” Also, in the following claims, the terms “including” and “comprising” are open-ended, that is, a system, device, article, composition, formulation, or process that includes elements in addition to those listed after such a term in a claim are still deemed to fall within the scope of that claim. Moreover, in the following claims, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects.

The above description is intended to be illustrative, and not restrictive. For example, the above-described examples (or one or more aspects thereof) may be used in combination with each other. Other embodiments can be used, such as by one of ordinary skill in the art upon reviewing the above description. The Abstract is provided to comply with 37 C.F.R. § 1.72(b), to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Also, in the above Detailed Description, various features may be grouped together to streamline the disclosure. This should not be interpreted as intending that an unclaimed disclosed feature is essential to any claim. Rather, inventive subject matter may lie in less than all features of a particular disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description as examples or embodiments, with each claim standing on its own as a separate embodiment, and it is contemplated that such embodiments can be combined with each other in various combinations or permutations. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. 

1. (canceled)
 2. A method of transferring molecules to cells of target tissue within a body of a patient comprising: delivering a plurality of molecules to the body of the patient; positioning at least one energy delivery body of an energy delivery device into the body of the patient within sufficient range of the target tissue so as to receive pulsed electric field energy delivered therefrom; and delivering the pulsed electric field energy to the at least one energy delivery body so as to transmit the pulsed electric field energy to the target tissue in a manner which causes at least one of the plurality of molecules to enter at least one of the cells of the target tissue, wherein the target tissue cells directly therapeutically benefit from the functionality of the molecules.
 3. A method as in claim 2, wherein directly therapeutically benefit comprises treatment of a disorder.
 4. A method as in claim 3, wherein the disorder comprises a genetic disorder.
 5. A method as in claim 2, wherein the at least one molecule of the plurality of molecules comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomer molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolysis targeting chimera, Zinc Finger Nucleases or Transcription Activator-Like Effector Nucleases, a protein and/or material which invokes genetic or epigenetic changes in cellular behavior.
 6. A method as in claim 2, wherein the at least one molecule of the plurality of molecules comprises a gene larger than 10 kb.
 7. A method as in claim 2, wherein delivering the plurality of molecules to the body comprises delivering the molecules intravenously to the body.
 8. A method as in claim 7, wherein delivering the plurality of molecules to the body comprises delivering the molecules intravenously to the body and locally to the target tissue.
 9. A method as in claim 2, wherein delivering the plurality of molecules occurs at least prior to delivering the energy.
 10. A method as in claim 2, wherein delivering the plurality of molecules comprises delivering the plurality of molecules to multiple locations within or near the target tissue.
 11. A method as in claim 10, wherein the multiple locations are within 0.5 mm-5 cm of the at least one cell of the target tissue.
 12. A method as in claim 10, wherein delivering the plurality of molecules at multiple locations comprises delivering different concentrations of molecule solution, different volumes of molecule solution and/or different types of molecule solution to one or more of the multiple locations.
 13. A method as in claim 10, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom, and wherein delivering the plurality of molecules at multiple locations is achieved by delivering molecules through one or more of the one or more tines.
 14. A method as in claim 13, wherein the delivering the plurality of molecules comprises delivering different molecules through at least one of the one or more tines in comparison to a least another of the one or more tines.
 15. A method as in claim 2, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom, and wherein the at least one energy delivery body comprises at least one of the one or more tines, and wherein delivering the energy comprises energizing at least one of the plurality of tines.
 16. A method as in claim 15, wherein energizing at least one of the plurality of tines comprises individually energizing at least one of the plurality of tines while at least one of the plurality of tines is not energized.
 17. A method as claim 2, further comprising inducing extravasation of fluid within a localized area in the body so as to increase delivery of molecules to the target tissue.
 18. A method as in claim 17, wherein inducing extravasation occurs prior to delivering energy.
 19. A method as in claim 17, wherein delivering the plurality of molecules comprises delivering the plurality of molecules to vasculature of the body and wherein the extravasation is from the vasculature.
 20. A method as in claim 17, wherein inducing extravasation comprises delivering conditioning energy to the at least one energy delivery body and wherein the conditioning energy is from another electric signal comprising a plurality of monophasic pulses each having a duration exceeding 500 microseconds.
 21. A method as in claim 2, further comprising delivering conditioning energy to the target tissue prior to delivering the pulsed electric field energy, wherein the conditioning energy increases cellular resistance of the target tissue to eventual cell death.
 22. A method as in claim 2, further comprising pre-warming the target tissue prior to delivering the pulsed electric field energy.
 23. A method as in claim 2, wherein the pulsed electric field energy is from an electric signal comprising a series of pulses, wherein the series of pulses includes at least one pulse having a positive amplitude and at least one pulse of having a negative amplitude.
 24. A method as in claim 23, wherein together the series of pulses have a balance of charge from positive amplitude on-time and negative amplitude on-time.
 25. A method as in claim 24, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid muscle stimulation within the body.
 26. A method as in claim 24, wherein together the series of pulses have sufficient balance of charge from positive amplitude on-time and negative amplitude on-time so as to avoid ablation of the target tissue cells. 